Immature Female Rats Research Articles

P-678 Cabergoline has inhibitory effects on FSH-induced cell differentiation and estrogen secretion in a primary culture system of rat ovarian granulosa cells

Abstract Study question How does cabergoline have effects on cell differentiation and hormone secretion induced by FSH stimulation in ovarian granulosa cells? Summary answer Cabergoline suppresses LH receptor expression and Estradiol (E2) secretion in primary cultures of rat ovarian granulosa cells by inhibiting cAMP production induced by FSH stimulation. What is known already Cabergoline is a dopamine receptor 2 agonist that has been used to treat hyperprolactinemia and, more recently, to prevent ovarian hyperstimulation syndrome. Although it has been previously reported that cabergoline suppresses E2 and Progesterone secretion induced by FSH stimulation in a primary culture system of rat ovarian granulosa cells, but the effect on LH receptor expression has not been investigated. It has also been reported that the activation of dopamine receptor 2 suppresses VEGF secretion and reduces the incidence of early-onset OHSS by inhibiting the VEGF/VEGFR2 pathway in research using human luteinized granulosa cells. Study design, size, duration We used a primary culture system of ovarian granulosa cells obtained from immature female rats treated with diethylstilbestrol to examine the effect of cabergoline addition on the changes in ovarian granulosa cells induced by FSH stimulation. Participants/materials, setting, methods FSH and cabergoline were added alone or co-added to a primary culture system of rat ovarian granulosa cells, and the changes induced by them were measured and compared. (1) mRNA expression of Lhcgr, Cyp19a1, and Fshr by RT-qPCR (2) E2 concentration in the culture medium (3) Measurement of hCG binding on the cell surface (4) cAMP concentration Main results and the role of chance Co-addition of cabergoline suppressed each of the changes induced by FSH stimulation as follows. (1) The mRNA expression levels of Lhcgr, Cyp19a1, and Fshr decreased in a concentration-dependent manner (0.01-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, they decreased to 14%, 9%, and 49% of FSH alone, respectively. (2) E2 concentration in the culture media also decreased in a concentration-dependent manner (1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 49% of FSH alone. (3) The amount of hCG binding on the cell surface also decreased in a concentration-dependent manner (0.1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 7% of FSH alone. (4) FSH-stimulated cAMP production decreased in a concentration-dependent manner (0.01-1µM) of co-added cabergoline compared to FSH alone. In the 1µM of co-added cabergoline group, it decreased to 50% of FSH alone. These results suggest that cabergoline suppresses LH receptor and aromatase expression and E2 production by inhibiting cAMP production induced by FSH stimulation. Limitations, reasons for caution Since this is an in vitro experiment using a rat primary culture system, the results of this research cannot be considered directly applicable to humans. Wider implications of the findings Our findings imply that cabergoline administration during the follicular phase may inhibit ovarian granulosa cell differentiation and estrogen secretion. In women taking relatively high doses of cabergoline, attention may need to be paid regarding the effects of cabergoline on follicular development and estrogen secretion. Trial registration number not applicable

P-081 A first in class potentiating therapeutic antibody as a new male fertility treatment

Abstract Study question Can we improve spermatogenesis in male by increasing FSH activity with a potentiating antibody? Summary answer IGYXOS developed a first in class potentiating therapeutic antibody that is able to stimulate spermatogenesis better than gonadotropins only as demonstrated in animal models. What is known already Idiopathic male infertility, including oligozoospermia, oligoasthenoteratospermia (OATS) and azoospermia, accounts for at least 75% of infertile men population, and no treatment is available at the moment for them. IGYXOS developed a monoclonal antibody directed against human FSH. IGX12 is a humanized IgG4 and it is able to potentiate FSH activity in vitro and in vivo in several animal models. Study design, size, duration IGX12 was tested in azoospermic rat and mouse models. The first model was created by treating Wistar Han rats with a GnRH antagonist. Gonadotropin treatment started after full inhibition of spermatogenesis. Four rats per group were treated with FSH+hCG regimen alone or in combination with IGX12. The second model was hpg mice that are congenitally hypogonadal because of their inability to synthetize GnRH. Seven mice per group were treated with FSH only or FSH+IGX12. Participants/materials, setting, methods IGX12 was tested in vitro on HEK293 cells and in vivo in Steelman and Pohley bioassay. The potentiating effect was investigated in azoospermic rats, that were treated for 8 weeks with FSH+hCG or FSH+hCG+IGX12. Hpg mice were treated with Gonal-f or Gonal-f+IGX12 for 7 weeks, followed by a treatment with Menopur or Menopur+IGX12 for a further 7 weeks. At the end of treatment, sperm counts in the testes and in the epididymides were assessed. Main results and the role of chance In vitro on HEK 293 cells expressing the human FSH receptor, IGX12 combined to FSH improved both the potency (EC50) and the efficacy (Emax) of FSH by increasing the cyclic AMP production at all tested FSH concentrations. The potentiating activity was confirmed in vivo in the Steelman and Pohley bioassay. Ovaries weight of immature female rats was increased significantly in the group treated with hCG/FSH+IGX12 compared to hCG/FSH only. To further analyze the potential benefit of IGX12 on male fertility, its effect was investigated in azoospermic rodent models. In rats with acquired azoospermia, treatment with FSH+hCG reached ∼50% of spermatogenesis of wild-type (WT) animals. Doubling the dose of gonadotropins did not improve their effect, whereas the addition of IGX12 on top of FSH+hCG treatment during one spermatogenesis cycle only (8 weeks) significantly improved sperm count in the testes and in the epididymides, reaching the level of WT animals. In hpg mice with congenital azoospermia, sperm count in the testis was 3.6 times higher and the sperm count in the epididymides was 6 times higher in the group treated with IGX12+FSH compared with FSH only (p < 0.05). In conclusion, IGX12 combined to a standard treatment stimulated spermatogenesis better than standard gonadotropin treatment. Limitations, reasons for caution After several proofs of concept and regulatory safety and toxicology evaluation in different animal models, IGX12 safety and tolerability is now assessed in a randomized, double blinded clinical phase I study on healthy men and women volunteers. Wider implications of the findings This first in class potentiating antibody IGX12 represents an innovative and promising new therapy to treat male patients with idiopathic infertility, especially with oligozoospermia. Trial registration number not applicable

P-654 A first in class potentiating therapeutic antibody as a new treatment for female fertility

Abstract Study question Can we improve controlled ovarian stimulation (COS) outcome in female by increasing FSH activity with a potentiating antibody? Summary answer IGYXOS developed a first in class potentiating antibody that improves COS treatments’ outcome by increasing FSH efficacy in vivo as demonstrated in non-human primates (NHP). What is known already For a COS in IVF cycles, FSH-containing preparations are injected to patients in order to obtain mature and fertilizable oocytes. However, the efficacy of these treatments is still limited and several attempts are needed before reaching a pregnancy and a live birth. One of the parameters that could be improved to avoid several attempts is the number of mature and thus fertilizable oocytes. It was previously demonstrated that breeding animals treated with gonadotropins could develop anti-gonadotropin antibodies that enhance their bioactivity instead of inhibiting it. These females had a high kidding rate and were hyperprolific. Study design, size, duration IGYXOS developed a monoclonal antibody (mAb) IGX12, a humanized IgG4, directed against human FSH. IGX12 was selected based on its ability to increase FSH activity in vitro and in vivo in rat., It was tested in mature cynomolgus female monkeysthat were treated as women undergoing ovarian stimulation for IVF with FSH alone or in combination with IGX12. Participants/materials, setting, methods IGX12 was tested in vitro on HEK 293 cells expressing the human FSH receptor. In vivo, the mAb was tested according to the Steelman and Pohley protocol. The potentiating effect was further investigated in NHP treated with mAb combined to a standard FSH treatment, under an agonist protocol. After hCG injection, oocytes were retrieved and their quality was analyzed. Blood was collected for hormonal assessments. Main results and the role of chance In vitro on HEK 293 cells expressing the human FSH receptor, IGX12 combined to FSH improves both the potency (EC50) and the efficacy (Emax) of FSH by increasing the cyclic AMP production at all tested FSH concentrations. The potentiating activity was confirmed in vivo in the Steelman and Pohley bioassay. Immature female rats received hCG+FSH alone or combined to mAb, twice a day, during 3 days. The animals were sacrificed the fourth day of treatment and the weight of the ovaries was compared. Ovaries weight was increased significantly in the group treated with hCG/FSH+IGX12 compared to hCG/FSH only. To further analyze the potential benefit of a IGX12 on human fertility, its effect was investigated in mature cynomolgus monkeys. IGX12 combined to a standard FSH treatment stimulated the follicular growth better than FSH only, and tripled the number of collected mature oocytes. Improved follicular growth correlated well with increased estradiol secretion during follicular phase and progesterone secretion during luteal phase. Limitations, reasons for caution After several proofs of concept and regulatory safety and toxicology evaluation in different animal models, IGX12 safety and tolerability is now assessed in a randomized, double blinded clinical phase I study on healthy men and women volunteers. Wider implications of the findings This first in class potentiating antibody IGX12 represents an innovative and promising new therapy to better stimulate patients with limited response to classical FSH treatments in order to improve the outcome of COS, and for normo-responders to avoid repetition of treatments. Trial registration number not applicable

Dynamics of gonadotropin and thienopyrimidine derivative TP03 effects on ovulation and ovarian steroidogenesis in Follimag-stimulated immature female rats

BACKGROUND: Gonadotropin preparations, particularly human chorionic gonadotropin (hCG), are commonly used to induce ovulation and treat reproductive disorders in women, albeit with associated side effects. Low-molecular-weight allosteric agonists of the luteinizing hormone receptor (LHR), such as thieno[2,3-d]pyrimidine derivatives, offer a potential alternative. AIM: This study aims to compare the effects of thieno[2,3-d]pyrimidine TP03 and hCG on ovarian weight, corpus luteum formation, and plasma levels of estradiol, progesterone, and luteinizing hormone in immature female rats pre-treated with Follimag®. It also examines their impact on ovarian gene expression related to LHR and steroidogenesis. MATERIALS AND METHODS: TP03 and hCG were administered 48 h after the Follimag® injection at a dose of 20 mg/kg (i.p.) and 15 IU/rat (s.c.), respectively. Parameters were assessed at 1, 2, 4, 8, 16, and 24 h after TP03 and hCG administration. Plasma hormone levels were measured via ELISA, and ovarian gene expression was analyzed using real-time PCR. RESULTS: TP03 increased ovarian weight, progesterone levels in the blood, and expression of steroidogenic genes encoding the cholesterol-transporting protein StAR and the cytochromes CYP11A1 and CYP17A1. TP03 also stimulated corpus luteum formation (16–24 h after treatment). The temporal dynamics of its stimulating effects were similar to those of hCG, although their magnitude was slightly inferior to those of gonadotropin. TP03-induced decrease in blood estradiol levels and aromatase gene expression in the ovaries was also more moderate. Unlike hCG, which suppressed LHR gene expression 8 h after treatment, TP03 maintained a high LHR gene expression, preserving ovarian sensitivity to endogenous luteinizing hormone. CONCLUSIONS: TP03 exhibits potential as an ovulation inducer with milder stimulating effects on ovarian steroidogenesis than hCG, which reduces the risks of developing ovarian hyperstimulation syndrome and resistance to gonadotropins.

Chemical characterization and response of house rats towards faecal odour of conspecifics

Repeated use of rodenticides for rodent pest management leads to the development of bait shyness and resistance in rodents. Therefore, using pheromones for rodent management could be a significant step forward. The present study was conducted to determine the response of house rats towards odour of faecal extracts of conspecifics of different age groups and sexes. Rats of different maturity groups and sexes were exposed to 20% faecal extract of mature and immature male and female rats. Results indicated that faecal extract of estrous stage mature females has a significant attractant/repellent effect on mature rats of opposite/same sex, respectively. Gas chromatography-linked mass spectrometry of the faecal extract of estrous female rats identified 62 compounds, of which 16 were already reported in other animals as pheromonal compounds and might be used in communication by house rats. In the future, these identified compounds can be used in a fixed proportion after testing their efficacy to develop a stable lureto increase the trappability and acceptability of antifertility agents and rodenticide-based baits for the management of house rats.

Gonadotropic Activity of Pituitary Extract of African Catfish, Clarias gariepinus, in Mammals using Immature Wistar Rats as Models.

The pituitary glands normally produce and stores gonadotropic hormones (GnH) that are responsible for ovulation and spermiation in animals. However, whether fish pituitary extracts can elicit same effects in treated animals need elucidation as a prelude to their practical usage in animals. The aim of this study was to investigate the oestrus-inducing potential of the pituitary gland extract of the Africa Catfish (Clarias gariepinus) in immature Wistar rats. The experiment involved the use of 18 immature female Wistar rats and 10 male catfish brood stocks with the use of six Wistar rats per groups as follows: Group A had human chorionic gonadotropin (hCG) treatment. Group B had only normal saline treatment as the control whereas Group C had the C. gariepinus pituitary extract administration to induce oestrus with treatments occurring twice six hours apart in each group. There was an obvious expression of visible signs of heat and the presence of uterine horn oedema with significant (p < 0.05) increase in reproductive tract weight and uterine width and length. However, only progesterone levels increased significantly (p < 0.05) in the hCG and the C. gariepinus pituitary extract treated groups compared to other assayed hormones. These results showed that C. gariepinus pituitary extract has the capacity to induce oestrus in Wistar rats because of its gonadotropic effects, which needs further investigations at higher doses and for longer exposure periods for possible oestrus induction and synchronization in higher mammals. Further favourable results could herald the possible patent of the catfish pituitary extract for either experimental or commercial use in mammals.

Differential effects of follicle-stimulating hormone glycoforms on the transcriptome profile of cultured rat granulosa cells as disclosed by RNA-seq.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: a. human pituitary FSH18/21 (hypo-glycosylated); b. human pituitary FSH24 (fully glycosylated); c. Equine FSH (eqFSH) (hypo-glycosylated); and d. Chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10-15 x 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent distinctly different regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways.

A metabolomics approach for the evaluation of Ficus benghalensis female in vivo reproductive effects relative to its metabolite fingerprint as determined via UPLC-MS and GC-MS

Ethnopharmacological relevanceFicus benghalensis, commonly known as Banyan Fig, is the national tree of India and its aerial roots are used traditionally to treat female reproductive disorders. However, despite this traditional use, no pharmacological evidence could be traced supporting this use. Additionally, no comprehensive metabolite profiling was reported for F. benghalensis aerial roots. Aim of the studyThis study attempts to justify biochemically the traditional use of F. benghalensis aerial roots in treatment of female reproductive disorders and in relation to its secondary metabolite profile. Materials and methodsTotal ethanol extract (TEE) and subfractions [petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and n-butanol (BUF] were prepared from air-dried powdered aerial roots of F. benghalensis. Detailed in-vivo investigation of the hormonal activity and action mechanism of the total ethanol extract and subfractions was carried out through evaluation of estrogenic and gonadotropic activities. The estrogenic activity was evaluated on ovariectomized immature female rats through estimating uterine weight, vaginal cornification and serum estradiol level along with histological examination of uteri. The gonadotropic activity was measured by assay of follicle stimulating hormone (FSH) and luteinizing hormone (LH) like activities. Total follicular and corpora lutea counts in immature female rats were used to determine FSH and LH like activities, respectively in addition to histological picture of the genitalia. Comprehensive non-targeted metabolite profiling was carried out for the TEE and subfractions using UPLC-HRMS in negative and positive ionization modes. UPLC-MS fingerprint was subjected to principal component analysis (PCA) and partial least squares analyses to correlate the bioactivities to specific chemical constituents in F. benghalensis different subfractions. GC-MS was further used for non-polar silylated fractions. ResultsResults revealed that only the non-polar PEF and CHF displayed moderate estrogenic and FSH-like activities but with no LH-like activity. Metabolites profiling via (UPLC-HRMS) and multivariate PCA analysis enabled identification and comparison of various chemical classes in F. benghalensis extract and fractions. The active non-polar fractions revealed nearly similar metabolites profile being composed of isoflavonoids, triterpenes, sterols, fatty acids and cyclic peptides. In contrast, polar fractions were more abundant in apocarotenoids, fatty acyl amides, hydroxybenzoates and hydroxycinnamates in addition to two lignans. PLS analysis revealed strong correlation between hydroxylated fatty acids and pyranoisoflavones with estrogenic and FSH-like activities. GC-MS analysis was further employed for non-polar fractions profiling revealing for their enrichment in fatty acids/esters, terpenes, organic acids and phenolics. ConclusionThis is the first study to rationalize the use of F. benghalensis aerial root traditionally in treatment of gynecological disorders, revealing that the petroleum ether and chloroform non-polar subfractions of F. benghalensis showed estrogenic and FSH-like activity with absence of LH-like activity. This biological activity could possibly be attributed to its metabolites profile of isoflavonoids, fatty acids, triterpenes, sterols and cyclic peptides identified via UPLC-MS and GC-MS techniques. Consequently, F. benghalensis aerial roots should be used with caution in traditional treatment of female infertility or other reproductive disorders.

Comparison of Steroidogenic and Ovulation-Inducing Effects of Orthosteric and Allosteric Agonists of Luteinizing Hormone/Chorionic Gonadotropin Receptor in Immature Female Rats.

Gonadotropins, including human chorionic gonadotropin (hCG), are used to induce ovulation, but they have a number of side effects, including ovarian hyperstimulation syndrome (OHSS). A possible alternative is allosteric luteinizing hormone (LH)/hCG receptor agonists, including the compound TP4/2 we developed, which remains active when administered orally. The aim was to study the effectiveness of TP4/2 (orally, 40 mg/kg) as an ovulation inducer in FSH-stimulated immature female rats, compared with hCG (s.c., 15 IU/rat). TP4/2 stimulated progesterone production and corpus luteum formation; time-dependently increased the ovarian expression of steroidogenic genes (Star, Cyp11a1, Cyp17a1) and genes involved in ovulation regulation (Adamts-1, Cox-2, Egr-1, Mt-1); and increased the content of metalloproteinase ADAMTS-1 in the ovaries. These effects were similar to those of hCG, although in some cases they were less pronounced. TP4/2, in contrast to hCG, maintained normal LH levels and increased the ovarian expression of the LH/hCG receptor gene, indicating preservation of ovarian sensitivity to LH, and did not cause a sustained increase in expression of vascular endothelial growth factor-A involved in OHSS. Thus, TP4/2 is an effective ovulation inducer that, unlike hCG, has a lower risk of OHSS and ovarian LH resistance due to its moderate stimulating effect on steroidogenesis.

Determination of biological activity of gonadotrophins in inbred and outbred animals. Part 1: Determination of biological activity of follicle-stimulating hormone

Scientific relevance. The biological activity of medicinal products may vary depending on the method of production (i.e. biological or recombinant products). The widening variety of gonadotrophin preparations, the diversity of their production methods, and the irreplaceability of biological activity bioassays with physicochemical tests require improvement of animal testing conditions.Aim. This study aimed to determine the biological activity of follicle-stimulating hormone (FSH) in several rat lines, analyse the findings, and select the most optimal testing conditions.Materials and methods. The biological activity was determined using in vivo methods. The comparative analysis used test results obtained over several years in inbred and outbred rats treated with FSH. In all cases, the authors used a three-dose randomised method based on the determination of the biological activity of test samples by comparison with that of the WHO international standard (IS) containing 183 IU of FSH bioactivity and 177 IU of LH bioactivity per ampoule (NIBSC code: 10/286). The study included immature female rats, inbred (Wistar-Kyoto or Sprague Dawley) and outbred. Testing conditions depended on the selected rat line, with the main variables being the test dose and the number of animals per group.Results. The authors compared responses of inbred and outbred rats to various doses of the FSH IS and test samples. Given the narrow range of the analytical procedure, Wistar-Kyoto rats showed a relatively weak dose–response relationship. The study demonstrated that the doses and testing duration depended on the sensitivity of the animals. Test results were less variable in inbred rats than in outbred ones. The statistical analysis of the results of FSH bioactivity testing in inbred and outbred rats showed that, with inbred rats, the number of animals could be halved without compromising the validity of the test.Conclusions. The approach proposed in this study provides for testing the biological activity of FSH with fewer experimental animals, improved cost-effectiveness, and the same reliability of results.

Життєздатність та види загибелі лейкоцитів крові щурів з синдромом полікістозних яєчників за умов переривчастого холодового впливу

The aim of our work is to assess the viability and determine the types of death of blood leukocytes in rats with polycystic ovary syndrome (PCOS) under conditions of intermittent cold exposure. The study was performed on 40 immature female rats of the WAG population, aged 27 days, divided into 5 groups of 8 animals each. Group 1 included intact rats. In group 2, rats were subcutaneously injected with 0.2 ml of purified and sterilized olive oil daily for 25 days. In group 3, rats were exposed to periodic cold exposure daily for 25 days. In group 4, dehydroepiandrosterone-induced PCOS was modeled in rats. In group 5, rats were injected with dehydroepiandrosterone against the background of periodic cold exposure. Leukocyte viability and death pathways were determined using a FACS Canto II flow cytometer (Becton Dickinson, USA). Rats with dehydroepiandrosteroneinduced PCOS showed a decrease in the percentage of viable leukocytes and an increase in the percentage of leukocytes in the early stage of apoptosis, while the number of leukocytes in the late stage of apoptosis and necrotic leukocytes did not change. In rats, on the background of the introduction of dehydroepiandrosterone under conditions of intermittent cold exposure, the percentage of viable leukocytes, as well as leukocytes at different stages of apoptosis and necrosis, corresponded to the physiological norm. Thus, the authors revealed a decrease in the viability of neutrophilic leukocytes in the blood of rats with dehydroepiandrosterone-induced PCOS due to the activation of apoptosis processes, as evidenced by an increase in the percentage of leukocytes at the initial stages of apoptosis. Intermittent cold exposure (4 h daily at +4°C for 25 days) against the background of dehydroepiandrosterone administration to rats had an anti-apoptotic effect, normalizing the viability of neutrophilic blood leukocytes.

Isolation, Characterization and Antifertility Activity of Ethanolic Extracts of Tabernaemontana divaricata R.Br. ex Roem. &amp; Schult

Because of the unequalled availability of chemical variety, natural products from medicinal plants, whether as pure compounds or as standardised extracts, provide limitless prospects for new therapeutic leads. The search for medicinal medications from natural products has increased the demand for chemical variety in screening programmes, which has led to a rise in interest in culinary plants in particular. Medicinal botanicals and herbal medicines contain a variety of bioactive chemicals. Worldwide, ethnic medical systems use Tabernaemontana divaricata (T. divaricata Apocynaceae) because of its significant therapeutic value. The goal of this work was to separate and identify the bioactive components of T. divaricata's ethanol fraction. Using two different experimental animal models, the isolated fraction's anti-fertility efficacy was assessed: 1) Ethinyl estradiol was used as the standard to measure the estrogenic activity in immature female rats. Modifications in uterine weight and uterine histology are among the evaluating criteria. 2) Female Wistar rats experienced anti-implantation and early abortifacient action. Using column chromatography and gradient elution with several mobile phases, the isolation was carried out. The isolated chemical was analysed using spectroscopy. On the basis of spectrum analyses (UV, IR, 1H NMR, and MASS), the structure was determined. The extracted substance from the ethanol fraction of the root was identified as 25-Methyl-25-norethyl-Stigmast-5-en-3-one and 19, 20-dimethyl-betulinic acid-3-yl-3'-hydroxyhexadecanoate based on spectrum characteristics. The uterine weight and height of stoma and epithelial cells in the estrogenic model are significantly higher than those in the ethanolic fraction of T. divaricata, however the tumour size is significantly smaller in the anti-implant model. This is the first time that the molecule 25-Methyl-25-norethyl-Stigmast-5-en-3-one and 19, 20-dimethyl-betulinic acid-3-yl-3'-hydroxyhexadecanoate from the ethanol fraction of T. divaricata roots has been reported. These compounds have potential as sources for the treatment of a number of disorders.&#x0D; Keywords: Tabernaemontana divaricata, Estrogenic activity, Anti-fertility effect, Anti-implantation activity, Abortifacient effect, Isolation

AGE AND SEX CHARACTERISTICS OF THE BEHAVIOR OF PRENATALLY STRESSED RATS IN A BLACK-AND-WHITE MODIFICATION OF THE BEHAVIORAL MODEL “SUOK-TEST”

Research activity, anxiety and motor disintegration were studied in prenatally stressed immature and adult male and female rats in the Suok-test model. The mothers of these animals from the 16th to the 19th days of pregnancy were placed for 3 hours in plastic pencil cases that fix the animals. The offspring were tested on day 20 and month 4 of postnatal ontogenesis. Mature females were tested taking into account the estrous cycle. A decrease in anxiety and an increase in research activity was observed in all experimental groups, with the exception of mature females at the diestrus stage. At this stage of the estrous cycle, behavioral patterns were identified in females that characterize anxiety and a decrease in the research component of behavior. Sensorimotor disintegration characteristic of immature animals was compensated with age, but not in females at the diestrus stage. Thus, according to the results of the study, it can be concluded that prenatal stress can carry both adaptive functions, causing the growth of the research component of behavior, and maladaptive ones. The anxiolytic effects of prenatal stress are rather negative, reducing evolutionarily significant protective mechanisms.

Characterization of the volatile profile and its estrogenic activity in Kadsura coccinea fruit.

The fruit of Kadsura coccinea (Lem.) A. C. Smith is an ethnomedicine used to treat abnormal menstruation, menopausal syndrome, and female infertility among the Dong Nationality in China. Our study aimed to identify the volatile oil profiles of the K. coccinea fruit and elucidate their estrogenic activity. The peel volatile oil (PeO), pulp volatile oil (PuO), and seed volatile oil (SeO) of K. coccinea were extracted using hydrodistillation and qualitatively analyzed using gas chromatography-mass spectrometry (GC-MS). Estrogenic activity was evaluated in vitro using cell assay and in vivo using immature female rats. Serum 17β-Estradiol (E2) and follicle-stimulating hormone (FSH) levels were detected using ELISA. In total, 46 PeO, 27 PuO, and 42 SeO components representing 89.96%, 90.19%, and 97% of the total composition, respectively, were identified. The compounds with the highest content in PeO, PuO, and SeO were β-caryophyllene, γ-amorphene, and n-hexadecanoic acid, respectively. PeO induced proliferation of MCF-7cells with an EC50 of 7.40μg/mL. Subcutaneous administration of 10mg/kg PeO significantly increased the weight of the uteri in immature female rats, with no effect on serum E2 and FSH levels. PeO acted as an agonist of ERα and ERβ. PuO and SeO showed no estrogenic activity. The chemical compositions of PeO, PuO, and SeO of K. coccinea are different. PeO is the main effective fraction for estrogenic activities, providing a new source of phytoestrogen for the treatment of menopausal symptoms.

Does cabergoline administration affect endometrial VEGFR-2 expression in a rat model of ovarian hyperstimulation syndrome?

Objective To assess the effect of cabergoline on endometrial vascular endothelial growth factor receptor-2 (VEGFR-2) immunoexpression in an ovarian hyperstimulation syndrome (OHSS) rat model. Material and Methods Twenty-one immature female Wistar rats were assigned into three groups: group 1, the control group; group 2, stimulated with gonadotropins to mimic OHSS; and group 3, in which an OHSS protocol was induced and thereafter treated with cabergoline (100 μg/kg/day). Body weight, ovarian volume, corpora lutea numbers, and endometrial VEGFR-2 expression were compared between the groups. Results Weight gain and ovarian volume were highest in the OHSS-placebo group, while cabergoline administration significantly reversed those effects (p = 0.001 and p = 0.001, respectively). VEGFR-2 stained cells were significantly lower in groups 2 and 3 compared to group 1 (p = 0.002). Although VEGFR-2 expression was lowest in group 3, the difference was not statistically significant. Corpora lutea numbers were also similar (p = 0.465). Conclusion While successful implantation requires a vascularized receptive endometrium, impaired expression of VEGFR-2 and disrupted endometrial angiogenesis due to cabergoline administration may be associated with IVF failure in fresh OHSS cycles. The insignificant decrease in endometrial VEGFR-2 expression observed in this research needs to be investigated by further studies involving additional techniques such as immunoblotting and/or RT-PCR analyses.

The investigation of cholinergic receptor muscarinic 1 activity in the rat ovary with induced ovarian hyperstimulation.

We look at the immunoreactivity of cholinergic receptor muscarinic 1 (CHRM1) in the ovarian tissues of rats with ovarian hyperstimulation syndrome (OHSS) considering the possibility that the muscarinic activity may contribute to the pathophysiology of OHSS. In this study, 14 immature female Wistar Albino rats were divided into two groups at random. The rats were 22 days old. Rats in the control group (n=7) were 22 days old, while those in the OHSS group (n=7) received 10 IU follicle-stimulating hormone subcutaneously over the course of four days and 30 IU human chorionic gonadotropin (hCG) on the fifth day to induce ovarian hyperstimulation. All the rats were sacrificed after all the groups' ovaries and blood samples were collected at the conclusion of the experiment. The left ovarian tissues were kept in aluminum foil at -80 °C, while the right ovarian tissues were kept in 10% formalin. Tissue vascular endothelial growth factor (VEGF), interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α and malondialdehyde (MDA) levels were measured by The Enzyme Linked Immunosorbent Assay technique in the ovarian tissues. CHRM1 immunoreactivity was scored immunohistochemically. Ovarian weight, tissue IL-10, TNF-α, VEGF and MDA levels, and CHRM1 immunoreactivity were significantly increased in the OHSS group. Increased levels of CHRM1 activity may play a role in the pathophysiology of OHSS. With further studies, the effect of luteinizing hormone and hCG on the ovarian and hypothalamic cholinergic system can be further investigated, and useful information can be obtained in determining OHSS prevention strategies.

Histological study to the effect of Potassium Dichromate on the reproductive organs in mature rats

This study was designed to show the effect of potassium dichromate (24 mg/kg orally for 60 day) in mature male and female. The results of experiment in male rats showed decrease in seminiferous epithelia thickness and number of stage VII cells (spermatogonium, spermatocytes, spermatids) at the same time the experiment of immature female rats treated with potassium dichromate showed increase in the diameter of ovarian follicles in the categories (101-200), (201-300) and (&lt;400) .It was concluded that potassium dichromate has detrimental effects on living tissue and in some sexual parts in both sexes (male and female) .

STIMULATION OF OVULATION IN IMMATURE FEMALE RATS USING ORTHOSTERIC AND ALLOSTERIC LUTEINIZING HORMONE RECEPTOR AGONISTS

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are widely used for the treatment of reproductive disorders and for controlled ovulation induction, but their use is limited by side effects. Allosteric agonists of the LH/hCG receptor, including thieno[2,3-d]thienopyrimidine TP03 developed by us, can become an alternative. TP03 (50 mg/rat, i.p.) when administered to immature female rats treated 48 hours before with Follimag has been shown to increase progesterone levels (maximum 8 hours post-treatment) and induce ovulation, as indicated by the appearance at 24 hours corpus luteum (8.6 ± 0.5 per ovary). In terms of its activity, TP03 is comparable to hCG, although it acts more moderately. In the ovaries, unlike hCG, TP03 does not lead to an increase in the expression of vascular endothelial growth factor, which can cause ovarian hyperstimulation syndrome. Thus, TP03 is a promising drug as an ovulation inducer and ovarian steroidogenesis stimulator.

REACTIVE OXYGEN SPECIES GENERATION BY BLOOD LEUCOCYTES OF RATS WITH POLYCYSTIC OVARY SYNDROME UNDER THE CONDITIONS OF INTERMITTENT COLD EXPOSURE.

The aim of this study was to determine the level of ROS production by blood leukocytes of rats with PCOS under the conditions of intermittent cold exposure. Materials and methods: In the study, 40 immature female rats of the WAG population at the age of 27 days with a body weight of 80-90 g were used. Five groups were formed (8 animals in each group). Group 1 was represented by intact rats that were not subjected to any manipulations. Group 2 was represented by rats that were injected subcutaneously with 0.2 ml of purified and sterilized olive oil daily for 25 days. Group 3 was represented by rats that were exposed to intermittent cold for 25 days. Group 4 was represented by rats that were modeled with PCOS. Group 5 was represented by rats, which were simulated PCOS against the background of intermittent cold exposure. ROS production was estimated in leukocytes isolated from rats of all groups by flow cytometry using the fluorescent probe of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA). Results: The experimental study revealed an intracellular excessive production of ROS by leukocytes in rats with polycystic ovary syndrome. The use of inter¬mittent cold exposure normalized the production of reactive oxygen species by leukocytes in rats with polycystic ovary syndrome. Conclusions: The effectiveness of intermittent cold exposure, proven by the authors, allows recommending its use as one of the methods of prevention and treatment of the polycystic ovary syndrome.

Stimulation of Ovulation in Immature Female Rats Using Orthosteric and Allosteric Luteinizing Hormone Receptor Agonists.

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) are widely used for the treatment of reproductive disorders and for controlled ovulation induction, but their use is limited by side effects. Allosteric agonists of the LH/hCG receptor, including thieno[2,3-d]thienopyrimidine TP03 developed by us, can become an alternative. TP03 (50 mg/rat, i.p.) when administered to immature female rats treated 48 h before with Follimag has been shown to increase progesterone levels (maximum 8 h post-treatment) and induce ovulation, as indicated by the appearance at 24 h corpus luteum (8.6 ± 0.5 per ovary). In terms of its activity, TP03 is comparable to hCG, although it acts more moderately. In the ovaries, unlike hCG, TP03 does not lead to an increase in the expression of vascular endothelial growth factor, which can cause ovarian hyperstimulation syndrome. Thus, TP03 is a promising drug as an ovulation inducer and ovarian steroidogenesis stimulator.