Gonadotropin Receptor Expression Research Articles

Exposure of antral follicles to medroxyprogesterone acetate during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed cycles.

Does medroxyprogesterone acetate (MPA) exposure in progestin-primed ovarian stimulation (PPOS) cycles cause molecular perturbations in the steroidogenic function and gonadotropin responsiveness of the granulosa cells? PPOS cycles are identical to traditional GnRH antagonist cycles not only for clinical IVF characteristics but also for gonadotropin receptor expression, response to gonadotropins, and steroidogenic function at the molecular level. PPOS is increasingly used as an alternative to GnRH antagonists due to the inhibitory effect of progesterone on LH release by reducing GnRH pulsatility at the hypothalamic level. Although a growing body of evidence from clinical studies did not indicate significant differences between PPOS and antagonist protocols for IVF cycle characteristics and obstetrical outcomes, it is still unknown whether exposure of the antral follicle cohort to progesterone or its synthetic derivatives during ovarian stimulation causes any subtle molecular aberrations in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed comparative molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from normal responding IVF patients undergoing PPOS and antagonist cycles. A clinical translational research study was conducted with IVF patients. This study included 55 normal responding IVF patients who underwent ovarian stimulation with either PPOS using MPA (5 mg twice daily) or GnRH antagonist cetrorelix acetate. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation triggering, respectively. Luteinized mural GCs obtained during the oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging, and hormone assays were used. Demographic and IVF cycle characteristics of the patients undergoing ovarian stimulation with PPOS and GnRH antagonist were similar, including ovarian response, mature oocyte yield, and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, SCC, 3β-HSD, 17β-HSD, aromatase) and the uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase, and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to those of the antagonist cycles. The expression of the receptors for gonadotropins, estrogen, and progesterone hormones was also similar. Basal and hCG-induced increases in 3β-HSD expression and progesterone production and basal and FSH-induced increases in aromatase expression and E2 output of the GCs from PPOS patients did not exhibit any meaningful differences when compared with GCs from antagonist cycles. Furthermore, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other important genes regulating cumulus expansion, ovulation, and luteal function [Relaxin, ADAMTS-1, and epidermal growth factor (EGF)-like growth factor amphiregulin] in the GCs of the PPOS and antagonist cycles were similar. N/A. Caution should be exercised when interpreting our data which was derived from normally responding patients whose ovulation was triggered with hCG. It is unclear whether the molecular parameters assessed vary according to infertility etiologies, magnitude of ovarian response, mode of trigger, and any other underlying ovarian pathologies or systemic diseases. MPA was the progestin used for PPOS and whether these findings can be generalized to other progestins is unknown. This study provides reassuring molecular evidence that exposure of antral follicle cohorts to MPA during the follicular growth phase does not have any detrimental effects on steroidogenic, ovulatory, and luteal functions when compared with GnRH antagonist cycles. This study was funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koç University Research Center for Translational Medicine (KUTTAM), and equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest. N/A.

Asprosin regulates male reproduction in teleosts: an in vitro study in Channa punctata.

The novel adipokine asprosin, produced by the furin enzymatic cleavage of profibrillin 1 protein (encoded by the Fbn1 gene), is implicated in regulating many physiological functions, including reproduction in mammals. In males, asprosin is reported to increase sperm density, sperm motility, and steroid production by interacting with Olfr734, which belongs to the G-protein-coupled receptor family (GPCR). In 2023, our group predicted and characterized asprosin in silico for the first time and demonstrated the robust expression of fbn1, and furin in the gonads of teleost spotted snakehead (ss) Channa punctata. Taking it forward, in the current study, we have investigated the effect of asprosin on the testicular functions of the spotted snakehead. As C. punctata is a seasonal breeder, the reproductive-phase-dependent expression of fbn1 in the testis was analyzed, showing significant upregulation during the preparatory and post-spawning phases. Additionally, bacterially overexpressed recombinant asprosin of C. punctata was purified to study the effect of ss asprosin on gametogenesis and steroidogenesis. Ex vivo treatment with recombinant asprosin resulted in significant upregulation of spermatogenic marker genes pcna, aldh1a2, cyp26a1, and sycp3. Asprosin also enhanced the gene expression of gonadotropin receptors, as well as sex steroid receptors, in addition to steroidogenic genes star and cyp17a1. Further exploring the downstream signaling cascade, the second messenger of GPCRs, cAMP levels following asprosin treatment were analyzed. Asprosin treatment prominently enhanced cAMP levels, thereby indicating the involvement of GPCR in the transduction of asprosin action. Hence, the study elucidates the regulation of male reproductive function by asprosin in spotted snakeheads.

Impact of Amphiregulin on Oocyte Maturation and Embryo Quality: Insights from Clinical and Molecular Perspectives

AbstractIdentifying non-invasive biomarkers which can predict the outcome of intracytoplasmic sperm injection (ICSI) is crucial, particularly in Germany where the challenges are intensified by the Embryo Protection Act. Recent research has highlighted biomarkers within the epidermal growth factor (EGF) family as central to follicular processes, although their predictive utility remains a subject of debate in the literature. Therefore, the primary objective of this study was to investigate the significance of amphiregulin concentrations in follicular fluid and gene expression in mural granulosa cells on oocyte maturation, fertilization, and embryo quality.A total of 33 women were recruited at the University Clinic of Saarland Fertility Center (Homburg, Germany). Follicular fluid aspiration consisted of single/individual aspiration of follicles, enabling a 1 : 1 correlation with retrieved oocytes. Follicular fluid and mural granulosa cell samples from 108 oocytes were analyzed. Amphiregulin levels were determined with enzyme-linked immunosorbent assay, while gene expression was analyzed with the StepOnePlus Real-Time PCR System using TaqMan Fast Advanced Master Mix assays.Results showed that amphiregulin concentrations affect oocyte maturation, fertilization, and embryo quality, while luteinizing hormone concentrations influence oocyte maturation, with significant differences identified between fertilized/unfertilized and good/poor embryo groups. Amphiregulin expression significantly impacts oocyte maturation, with downregulation observed in immature oocytes, while luteinizing hormone/chorionic gonadotropin receptor expression showed no significant differences between groups and did not influence maturation, fertilization, or embryo quality.These findings are very important for advancing infertility treatment, especially in Germany. The results for amphiregulin may provide prognostic insights which could be useful when selecting viable oocytes and embryos. This research underscores the importance of non-invasive biomarkers for optimizing ICSI outcomes and potentially enhancing the success rates of assisted reproductive technology.

O-299 Exposure of antral follicles to progestins during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed IVF cycles

Abstract Study question Does progestin exposure cause any molecular perturbations in the steroidogenic features and gonadotropin responsiveness of the granulosa cells in progestin-primed ovarian stimulation (PPOS) cycles? Summary answer No. PPOS cycles are identical to antagonist cycles for gonadotropin receptor expression, response to gonadotropins, steroidogenic function at molecular level in normal responding IVF patients. What is known already PPOS is commonly used in IVF cycles as an alternative to antagonist cycles. Although accumulating evidence from clinical studies did not indicate any particular differences between these two types of stimulation protocol for IVF cycle characteristics, it is still unknown if exposure of antral follicle cohorts to synthetic progesterone derivatives during ovarian stimulation causes any subtle molecular aberrations in PPOS cycles in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from the normal responding patients undergoing PPOS vs. antagonist IVF cycles. Study design, size, duration A translational research study in IVF patients. Participants/materials, setting, methods 55 normal responding IVF patients underwent ovarian stimulation either with PPOS using medroxy progesterone acetate (5mg twice daily) or, with GnRH antagonist cetrorelix acetate were included in the study. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation trigger, respectively. Luteinized mural granulosa cells obtained during oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging and hormone assays were used. Main results and the role of chance Demographic and IVF characteristics of the patients undergoing ovarian stimulation with PPOS vs. GnRH antagonist were similar including ovarian-response, mature oocyte yield and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, aromatase, 3B-HSD, 17B-HSD) and uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to the antagonist IVF cycles. Both cycles were also similar for the expression of FSH and LH receptors at transcriptional and translational level. Basal and hCG-induced increases in 3B-HSD expression and P4 production, and basal and FSH-induced increases in aromatase expression and E2 output of the GCs of the PPOS patients did not show any meaningful differences from the antagonist cycles. Similarly, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake rate did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other genes regulating cumulus expansion, ovulation and luteal function (Relaxin, ADAMTS-1 and EGF-like growth factor amphiregulin (AREG)) in the GCs of the PPOS cycles were comparable to the antagonist cycles. Limitations, reasons for caution Caution should be exercised when interpreting our data, which was produced in normal responders. It is unclear if the molecular parameters assessed here vary according to infertility etiologies, ovarian response type, mode of trigger and any other underlying ovarian pathology or systemic diseases that are concomitantly present. Wider implications of the findings This study provides reassuring molecular evidence that exposure of antral cohorts to progestins follicular growth phase does not appear to have any detrimental effect on their steroidogenic, ovulatory and luteal functions, suggesting that PPOS IVF cycles are not different from traditional antagonist cycles. Trial registration number not applicable

Patients with gynecological malignancies are similar to other IVF patients without cancer for clinical and molecular reproductive parameters and DNA damage response pattern

This study intended to investigate if gynecological cancers compromise ovarian function and reduce the success of assisted reproduction techniques (ART). No clinical and molecular data together is available on this issue for gynecological or other organ cancers. Steroidogenic pathways and DNA damage response characteristics of the granulosa cells retrieved from the 39 gynecological cancer patients were analyzed together with their clinical ART characteristics in comparison to 31 control ART patients. Patients with gynecological malignancies were similar to the control IVF patients for the number of mature oocytes retrieved, fertilization rates and embryo development competency. Molecular analyses of the granulosa cells retrieved from these cancer patients did not detect any perturbations in gonadotropin receptor expression and response, sex steroid production, cholesterol utilization/storage and, DNA damage response pattern in comparison to control IVF patients without cancer. This study provides the first reassuring clinical and molecular combined data set that the presence of gynecological malignancy does not appear to have any detrimental effect on clinical IVF cycle characteristics and ovarian functioning at molecular level.

Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro

AbstractKisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2) and progesterone (P4) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6–10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3β‐HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal‐regulated kinase 1/2, ERK1/2 and p‐ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p < .05), whereas P4 production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p‐ERK1/2 was observed in the Kp‐treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp‐mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Gonadotropin Receptor Cross-Talk and Altered Functions in Gonadal and Non-Gonadal Tissues

Reproduction depends on the responses of gonadotropins through their specific receptors. The gonadotropin family has three members; Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), and Human Chorionic Gonadotropin (hCG). These glycoprotein hormones comprise two subunits, an identical α-subunit and a hormone-specific-β subunit. Their cognate receptors (FSHR and LHCGR) are two adrenergic receptor-like family A/rhodopsin-like G-Protein Coupled Receptors (GPCRs) with structurally distinct ligand binding domains. The hCG binds to LHCGR but has a longer half-life and higher affinity to LHCGR. The expression of FSHR and LHCGR is observed in both gonadal and nongonadal cells. In this review, we will be emphasizing the differential expression of gonadotropin receptors in different cells of the human body, their specific responses through cross-talk, and how a defect in the expression and activity of FSHR and LHCGR may alter the responses of FSH and LH/hCG leading to diseases like PCOS, cancer and metabolic disorders.

Gene expression by equine oocytes and cumulus cells, a window into the unknown

In vitro maturation (IVM) of oocytes (OC) is a critical step for the in vitro production of equine embryos. However, the regulatory mechanisms, biological processes, cellular components, and molecular functions involved in IVM are not entirely understood. We previously analyzed the gene expression dynamics of gonadotropin receptors in cumulus cells (CC) during IVM, demonstrating an upregulation of the follicle-stimulating hormone receptor gene (FSHR) and downregulation of the luteinizing hormone choriogonadotropin receptor gene (LHCGR) in mature compared to immature CC (de la Fuente.et.al. Journal of Equine Veterinary Science 2022; 113). Moreover, we found that CC from in vivo maturing cumulus-oocyte complexes (COC) obtained from dominant gonadotropin-stimulated follicles (DF-CC) had a lower expression of FSHR and a higher expression of LHCGR compared to their in vitro counterparts. However, the effect of IVM medium composition on the expression of gonadotropin receptors has not been investigated. Thus, the aim of this study was to determine differences in the gene expression of OC and CC following IVM of three reported IVM media: A) EQ-IVM (IVF-Bioscience) a commercially available medium, B) DMEM/F12-based medium (Meyers.et.al. Reproduction, Fertility and Development. 2019; 31:1874-1884.) and C) M199-based medium (Choi.et.al. Theriogenology. 2002; 58:771-774) and their difference with DF-CC samples. Extracted total RNA from OC and CC was sequenced and raw reads were trimmed (trimmgalore) and mapped (STAR) to the current equine referencegenome. Mapped reads were quantified (featurecount) and compared between groups, using the DESeq2 package (FDR cutoff 0.1; Min (log2) fold change 1.5). Overall, there was a mapping rate of >85%, and an average of 14,000 expressed genes in OC and CC samples with ∼68% categorized as protein-coding genes. Based on gene expression dis-similarities, OC and CC samples were partially clustered according to their respective maturation media, but all samples showed a different gene expression profile compared to DF-CC samples. Comparing the gene expression of CC between the A and B media, we found 607 differentially expressed genes (DEGs), 1905 DEGs between A and C, and 957 DEGs between B and C. All three media showed a similar DEGs number when compared to DF-CC samples (∼10K DEGs). FSHR showed a higher expression in all IVM samples compared to DF-CC. However, LHCGR presented a lower expression only in two of the IVM media used (B & C) compared to DF-CC, showing no significant expression difference in the commercial IVM media tested. These data highlight the dynamics of gonadotropin receptors during IVM, which may contribute to the understanding of the physiology of oocyte maturation and allow optimization of IVM conditions in horses. Funding: The Theriogenology Foundation, the Population Health and Reproduction Department Seed Grant, and the Center for Equine Health.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

Melatonin administration during the first half of pregnancy improves the reproductive performance of rabbits: Emphasis on ovarian and placental functions

This study was designed to investigate the roles of melatonin administration during different sensitive windows of the first half of pregnancy in the function and gene expression of the ovary and placenta, hormone profile, and pregnancy outcomes in rabbits. Four equal experimental groups of 20 rabbits each were used. The first (FW), second (SW), and third (F + SW) groups comprised rabbits that orally received 0.7-mg melatonin/kg body weight during the first week, second weeks, and during both weeks of pregnancy; and the fourth group served as the control group (C). The total number of visible follicles significantly increased in all melatonin-treated groups compared with that in the C group. In all melatonin-treated groups, the number of absorbed fetuses was significantly reduced, whereas the weights of embryonic sacs and fetuses were higher than in the C group. The placenta efficiency was significantly increased in the F + SW group compared with that in the C group, followed by the SW group, whereas no significant difference in the placenta efficiency was found between the FW and C groups. Melatonin treatments significantly improved the expression of antioxidants, gonadotropin receptors, and cell cycle regulatory genes in the ovary, whereas only FW treatment upregulated steroidogenic acute regulatory gene. Compared with the C and FW groups, melatonin treatments during the SW and F + SW significantly upregulated the expression of most genes in the placenta. The concentrations of estradiol were significantly higher in the SW and F + SW groups than in the FW and C groups. The concentrations of progesterone were significantly increased in the FW group compared with those in the C and SW groups, whereas the F + SW group showed intermediate values. The litter size and weight at birth significantly increased in all melatonin-treated groups compared with those in the C group. The second week of pregnancy seems to be a sensitive window for melatonin actions during pregnancy. Thus, melatonin administration during the second week of pregnancy can be effective in improving pregnancy outcomes in rabbits.

Growth hormone in fertility and infertility: Mechanisms of action and clinical applications

Secreted by the anterior pituitary gland, growth hormone (GH) is a peptide that plays a critical role in regulating cell growth, development, and metabolism in multiple targeted tissues. Studies have shown that GH and its functional receptor are also expressed in the female reproductive system, including the ovaries and uterus. The experimental data suggest putative roles for GH and insulin-like growth factor 1 (IGF-1, induced by GH activity) signaling in the direct control of multiple reproductive functions, including activation of primordial follicles, folliculogenesis, ovarian steroidogenesis, oocyte maturation, and embryo implantation. In addition, GH enhances granulosa cell responsiveness to gonadotropin by upregulating the expression of gonadotropin receptors (follicle-stimulating hormone receptor and luteinizing hormone receptor), indicating crosstalk between this ovarian regulator and the endocrine signaling system. Notably, natural gene mutation of GH and the age-related decline in GH levels may have a detrimental effect on female reproductive function, leading to several reproductive pathologies, such as diminished ovarian reserve, poor ovarian response during assisted reproductive technology (ART), and implantation failure. Association studies using clinical samples showed that mature GH peptide is present in human follicular fluid, and the concentration of GH in this fluid is positively correlated with oocyte quality and the subsequent embryo morphology and cleavage rate. Furthermore, the results obtained from animal experiments and human samples indicate that supplementation with GH in the in vitro culture system increases steroid hormone production, prevents cell apoptosis, and enhances oocyte maturation and embryo quality. The uterine endometrium is another GH target site, as GH promotes endometrial receptivity and pregnancy by facilitating the implantation process, and the targeted depletion of GH receptors in mice results in fewer uterine implantation sites. Although still controversial, the administration of GH during ovarian stimulation alleviates age-related decreases in ART efficiency, including the number of oocytes retrieved, fertilization rate, embryo quality, implantation rate, pregnancy rate, and live birth rate, especially in patients with poor ovarian response and recurrent implantation failure.

IVF characteristics and the molecular luteal features of random start IVF cycles are not different from conventional cycles in cancer patients.

Are the IVF parameters and the steroidogenic luteal characteristics of random-start IVF cycles different from conventional cycles in cancer patients? No; controlled ovarian stimulation cycles randomly started at late follicular phase (LFP) and luteal phase (LP) are totally comparable to those conventional IVF cycles started at early follicular phase (EFP) in terms of the expression of the enzymes involved in cholesterol utilization and steroid hormone biosynthesis pathways, gonadotropin receptor expression and, estradiol (E2) and progesterone (P4) production in addition to the similarities in ovarian response to gonadotropin stimulation, oocyte yield, fertilization rate and embryo development competency in cancer patients. Random start ovarian stimulation protocols are commonly employed for oocyte and embryo freezing for fertility preservation in cancer patients with time constraints who do not have sufficient time to undergo ovarian stimulation initiated conventionally at EFP of the next cycle. No data is available regarding the molecular steroidogenic features of these cycles analyzed together with the clinical IVF characteristics in cancer patients. We aimed to address this question in this study to help understand how similar the random start cycles are to the conventional start ones. A clinical translational research study conducted in 62 cancer patients undergoing IVF for fertility preservation between the years 2017 and 2022. Sixty-two patients who were diagnosed with different types of cancer and underwent ovarian stimulation for oocyte (n = 41) and embryo (n = 21) cryopreservation using GnRH antagonist protocol and human menopausal gonadotropins before receiving cancer treatment/surgery were enrolled in the study. For patients with breast cancer and endometrial cancer the aromatase inhibitor letrozole was used with gonadotropin stimulation. Ovarian stimulation was initiated conventionally at EFP in 22 patients and served as control while it was started at LFP in 20, and mid-LP in the other 20 patients. The luteinized granulosa cells (GCs) were recovered from follicular aspirates during oocyte retrieval procedure and used for the experiments separately for each individual patient. The expression of the enzymes involved in sex steroid biosynthesis (StAR, 3β-HSD, Aromatase) and cholesterol synthesis (3-hydroxy 3-methylglutaryl Co-A reductase (HMG-Co-A reductase)), utilization (hormone sensitive lipase (HSL)), and storage (Acetyl-Coenzyme A acetyltransferase 1 (ACAT-1)), and gonadotropin receptor expression status were analyzed using immunoblotting and RT-PCR methods. Laser confocal immunofluorescence imaging was applied to analyze and compare the expression patterns of the steroidogenic enzymes and their relation with mitochondria. In vitro E2 and P4 production by the cells were compared among the groups. Baseline demographic and IVF characteristics of the patients undergoing the conventional start and random start IVF cycles were similar. Duration of gonadotropin stimulation was significantly longer in LFP and LP start cycles in comparison to the conventional ones. Ovarian response to gonadotropin stimulation, mature and total oocyte yield, fertilization and Day 5 blastulation rates of the embryos were comparable between the conventional versus random start cycles. When the luteal GCs of these random start cycles were analyzed we could not find any gross differences between these cycles in terms of the viability index and gross light microscopic morphologic features. More detailed analysis of the molecular luteal characteristics of the cells using RT-PCR, immunoblotting methods revealed that the expression profiles of the gonadotropin receptors, and the enzymes involved in sex steroid biosynthesis and cholesterol synthesis/utilization, and the steroidogenic activity of the luteal GCs of the random start cycles are almost identical to those of the conventional start cycles. Confocal image analysis demonstrated similar patterns in the signal expression profiles of the steroidogenic enzymes and their co-localization within mitochondria. N/A. Caution should be exercised when interpreting our data and counseling cancer patients seeking fertility preservation because it is still unclear if previous exposure to cancer drugs, different ovarian pathologies or infertility etiologies, previous ovarian surgery and/or any other underlying diseases that are concomitantly present with cancer may cause a difference between conventional and random start stimulation protocols in terms of IVF parameters, luteal function and reproductive outcome. Relatively low number of patients in each stimulation protocol and pooling of luteal GCs for each patient rather than individual analysis of each follicle and oocyte are additional limitations of our study. Our findings provide reassurance that random start protocol offers cancer patients an equally good prospect of fertility preservation as conventional IVF. Funded by the School of Medicine, the Graduate School of Health Sciences of Koc University and Koç University Research Center for Translational Medicine (KUTTAM), equally funded by the Republic of Turkey Ministry of Development Research Infrastructure Support Program. All authors declare no conflict of interest. N/A.

Prolactin promotes parental responses and alters reproductive axis gene expression, but not courtship behaviors, in both sexes of a biparental bird

Prolactin, a hormone involved in vertebrate parental care, is hypothesized to inhibit reproductive hypothalamic-pituitary-gonadal (HPG) axis activity during parenting, thus maintaining investment in the current brood as opposed to new reproductive efforts. While prolactin underlies many parental behaviors in birds, its effects on other reproductive behaviors, such as courtship, remain unstudied. How prolactin affects neuropeptide and hormone receptor expression across the avian HPG axis also remains unknown. To address these questions, we administered ovine prolactin (oPRL) or a vehicle control to both sexes in experienced pairs of the biparental rock dove (Columba livia), after nest removal at the end of incubation. We found that oPRL promoted parental responses to novel chicks and stimulated crop growth compared to controls, consistent with other studies. However, we found that neither courtship behaviors, copulation rates nor pair maintenance differed with oPRL treatment. Across the HPG, we found oPRL had little effect on gene expression in hypothalamic nuclei, but increased expression of FSHB and hypothalamic hormone receptor genes in the pituitary. In the gonads, oPRL increased testes size and gonadotropin receptor expression, but did not affect ovarian state or small white follicle gene expression. However, the oviducts of oPRL-treated females were smaller and had lower estrogen receptor expression compared with controls. Our results highlight that some species, especially those that show multiple brooding, may continue to express mating behavior despite elevated prolactin. Thus, mechanisms may exist for prolactin to promote investment in parental care without concurrent inhibition of reproductive function or HPG axis activity.

Partially purified non-polar phytocomponents from Aloe barbadensis Mill. gel restores metabolic and reproductive comorbidities in letrozole-induced polycystic ovary syndrome rodent model- an “in-vivo” study

Ethnopharmacological relevanceIn India, Kumaryasava, a popular Aloe barbadensis Mill. gel preparation has therapeutic value in treatment of female reproductive disorders like menstrual disturbances and menopausal problems. Despite their widespread use, only a limited number of studies have probed into the scientific evidence for their varied bioactivities. In this regard, studies have demonstrated that Aloe vera gel has the potential to modulate steroidogenic activity in letrozole induced polycystic ovary syndrome (PCOS) rat. However, isolation and identification of the bioactive molecule/s from Aloe vera gel and studying their molecular targets will underpin the treatment regime for PCOS. Material and methodsThe Partially Purified Non-Polar Phytocomponents (PPNPP)- LP1 and LP3 were isolated from the petroleum ether extract of Aloe vera gel by column chromatography. Based upon the GC-MS analysis, LP1 and LP3 comprised of n-Hexadecanoic acid and Campesterol acetate with an abundance of 97.07%, and 96.07% respectively. For evaluation of their bioactivities, eighty 3–4 months female Balb/c mice were classified as 10 groups with 8 animals in each group. Groups were control (C), PCOS (0.5 mg/kg/day Letrozole orally for 21days), PCOS treated orally for 60 days with Aloe vera gel (AVG) (10 mg/kg/day) (PCOS + AVG), PCOS treated orally for 60 days with petroleum ether extract (PE) of Aloe vera gel (25 μg/kg/day) (PCOS + PE), PCOS treated orally for 60 days with LP1 (0.5 μg/kg/day) (PCOS + LP1), PCOS treated orally for 60 days with commercially available pure compound-n-Hexadecanoic acid (HA) (0.5 μg/kg/day) (PCOS + HA), PCOS treated orally for 60 days with LP3 (0.01 μg/kg/day) (PCOS + LP3), PCOS treated orally for 60 days with commercially available pure compound- Campesterol acetate (CA) (0.01 μg/kg/day) (PCOS + CA), PCOS treated orally for 60 days with Metformin (100 mg/kg/day) (PCOS + Metformin) and PCOS treated orally for 60 days with DMSO (Vehicle) (PCOS + DMSO). Body weight, Oral glucose tolerance test, lipid profile, fasting glucose, insulin, estrus cycle, hormonal profile, gene expression of gonadotropin receptors (Fshr and Lhr), steroid receptors (Ar, Esr1, Esr2 and Pgr) and steroidogenic markers (Star, Hsd3b1, Cyp19a1 and Amh) were analysed in the ovaries. Polycystic ovarian morphology was assessed through histopathological changes of ovary. Toxicity markers- SGOT, SGPT and creatinine were also measured at the end of the study. ResultsMice treated with letrozole demonstrated significant increase in body weight, glucose intolerance, fasting insulin levels, HOMA-IR, triglycerides levels as well as testosterone levels, and a significant decline in the progesterone levels as compared to the control animals. PCOS animals also exhibited arrested estrus cyclicity, disrupted ovarian histopathology with the presence of multiple peripheral cysts and abnormal gene expression of gonadotropin receptor, steroid receptor and steroid markers. Oral administration of AVG, PE extract of AVG, LP3 and metformin greatly alleviated these complications in PCOS animals. ConclusionThe above findings indicate the effectiveness of LP3, isolated from Aloe vera gel against letrozole induced PCOS in mice and may be used in the treatment of PCOS as an alternative to metformin.

Sodium Fluoride In Vitro Treatment Affects the Expression of Gonadotropin and Steroid Hormone Receptors in Chicken Embryonic Gonads.

Simple SummaryEffects of in vitro sodium fluoride (NaF) treatment on the mRNA expression of luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), estrogen receptors (ESR1 and ESR2), progesterone receptor (PGR), and the immunolocalization of PGRs were examined in gonads of 14-day-old chicken embryos. In the ovary, the NaF treatment significantly increased mRNA levels of all investigated receptors. In the testes, the lowest applied dose of NaF (1.7 mM) significantly decreased the expression of FSHR, ESR1, ESR2, and PGR. Alternatively, the higher NaF dose (7.1 mM) elevated PGR mRNA level in the male gonad. Immunohistochemical analysis revealed that the NaF exposure increased PGR expression in the ovarian cortex, while it decreased its expression in the testes. Collectively, these data indicate that: (i) NaF may disturb the chicken embryonic development, and (ii) different mechanisms of this toxicant action exist within the female and male gonads.Sodium fluoride (NaF), in addition to preventing dental decay may negatively affect the body. The aim of this study was to examine the effect of a 6 h in vitro treatment of gonads isolated from 14-day-old chicken embryos with NaF at doses of 1.7 (D1), 3.5 (D2), 7.1 (D3), and 14.2 mM (D4). The mRNA expression of luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), estrogen receptors (ESR1 and ESR2), progesterone receptor (PGR), and the immunolocalization of progesterone receptors were examined in the tissue. In the ovary, the expression of FSHR and LHR increased following the NaF treatment. In the case of FSHR the highest stimulatory effect was noticed in the D2 group, while the expression of LHR increased in a dose-dependent manner. A gradual increase in ESR1 and PGR mRNA levels was also observed in the ovary following the NaF treatment, but only up to the D3 dose of NaF. The highest ESR2 level was also found in the D3 group. In the testes, the lowest dose of NaF significantly decreased the expression of FSHR, ESR1, ESR2, and PGR. On the other hand, an increase in PGR expression was observed in the D3 group. The expression of LHR in the testes was not affected by the NaF treatment. Immunohistochemical analysis showed that NaF exposure increased progesterone receptor expression in the ovarian cortex, while it decreased its expression in the testes. These results reveal that NaF may disturb the chicken embryonic development and different mechanisms of this toxicant action exist within the females and males.

Igf3: a novel player in fish reproduction†.

As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.

Impact of Serum Human Chorionic Gonadotropin and Luteinizing Hormone Receptor Expression to Oocyte Maturation Rate: A Study of Controlled Ovarian Stimulation.

Background:Few studies have assessed the impact of serum human chorionic gonadotropin (hCG) levels before oocyte retrieval and luteinizing hormone receptor (LHR) mRNA expression.Aims:The objective was to assess the correlations between serum hCG levels at 12-h posttrigger granulosa cell LHR mRNA expression during the in vitro fertilization (IVF) cycle with oocyte maturation rate and to determine the cutoff level of serum hCG at 12-h posttrigger.Settings and Design:A cross-sectional was conducted for this study at the IVF center of Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia.Materials and Methods:Recombinant follicle-stimulating hormone was used on day 2 of the menstrual cycle with multiple doses of a gonadotropin-releasing hormone antagonist. Recombinant hCG was used to trigger ovulation. At 12-h posttrigger, hCG serum levels were measured using an enzyme-linked immunosorbent assay.Statistical Analysis:Pearson's correlation coefficient was used to evaluate the correlation between oocyte maturation rates, serum hCG levels, and LHR mRNA levels. Cutoff values were determined using a receiver operating characteristic (ROC) curve.Results:Serum hCG levels were positively correlated (r = 0.467;P < 0.01), and LHR mRNA expression was weakly correlated (r = 0.073; P = 0.701) with oocyte maturation. The cutoff of serum hCG for a high maturation rate was 77 mIU/mL, with an area under the ROC curve of 0.765 (95% confidence interval: 0.598–0.939) andP < 0.001.Conclusion:Oocyte maturation is correlated with serum hCG levels with 77 mIU/mL as the cutoff point for oocyte retrieval.

Ontogeny of the specificity of gonadotropin receptors and gene expression in carp.

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are the principle endocrine drivers of reproductive processes in the gonads of jawed vertebrates. Canonically, FSH recruits and maintains selected ovarian follicles for maturation and LH induces the stages of germinal vesicle breakdown and ovulation. In mammals, LH and FSH specifically activate cognate G-protein-coupled receptors that affect the proteins involved in steroidogenesis, protein hormone synthesis, and gametogenesis. This dual-gonadotropin model also exists in some fish species, but not in all. In fact, due to their diverse number of species, extended number of ecological niches, and remarkably flexible reproductive strategies, fish are appropriate as models to understand the co-evolution of gonadotropins and their receptors. In this study, we cloned and characterized the expression profile over the final stages of ovarian maturation of carp (Cyprinus carpio) LHCGR and FSHR. Expression of both gonadotropin receptors increased in the later stage of early vitellogenesis, suggesting that both LH and FSH play a role in the development of mature follicles. We additionally tested the activation of cLHCGR and cFSHR using homologous and heterologous recombinant gonadotropins in order to gain insight into an evolutionary model of permissive gonadotropin receptor function. These data suggest that carp (Cyprinus carpio) gonad development and maturation depends on a specific gonadotropin profile that does not reflect the temporally distinct dual-gonadotropin model observed in salmonids or mammals, and that permissive gonadotropin receptor activation is a specific feature of Ostariophysi, not all teleosts.

Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

Estradiol-17β (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.

Pre-pubertal exposure to high temperature impairs ovarian and adrenal gland function in female rats.

The influence of different levels of heat exposure on the functions of ovarian and adrenal gland were investigated in pre-puberty female rats. Three-week old female rats were treated with control (26°C) or three higher temperatures (38, 40 and 42°C) for 2hr/day. After 9 days of treatment, blood samples were collected for measurement of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol-17β, corticosterone, cholesterol and triglyceride. Adrenal glands, ovaries and liver were collected for analyzing gene expressions. Body and liver weight were significantly low in the 42°C heating group. Circulating LH and triglyceride in the 42°C heating group were significantly lower, and estradiol-17β, corticosterone and cholesterol were significantly higher than those of the control group. The gene expression of 3β-HSD and P450c21 in the adrenal gland; 3β-HSD, receptors of LH, FSH and estrogen in the ovary were significantly low in heated rats. The liver gene expressions of caspase 3 and NK-κB were significantly high in 42°C heated rats, suggesting that the ability of liver metabolic function reduced in the 42°C heated rats. These results demonstrated that the high temperature is responsible for suppression of ovarian function by decreasing the expression of steroidogenic enzymes, estrogen and gonadotropin receptors in the ovary. Increase in circulating estradiol-17β in the heated rats may be due to accumulate this hormone in circulation by potential changes in liver metabolism during the heat stress.