The effect of the insulin-sensitizing drug metformin on preovulatory follicle (POF) number, ovulation rate, fetal rate and prolificacy was studied in forty-six cyclic Malpura ewes. After estrus synchronization, the ewes were equally divided into two groups (n = 23). The treatment group (MET) received a daily oral dose of metformin at a rate of 500 mg/animal for approximately 12 weeks, spanning five estrous cycles, as against untreated control (CON). All the ewes were bred to proven rams at the end of treatment. Ovarian ultrasound scans were performed at each estrus and day 9 of each cycle to assess the number and diameter of POFs and corpora lutea (CL), respectively. A comprehensive assessment of circulating hormones including, estradiol, progesterone, androstenedione, and insulin as well as metabolic indicators such as glucose, and lipid profile parameters was performed. At the end of treatment on the day of estrus (E5D0), the treatment showed a stimulatory effect on follicular development with a 53.2% (P < 0.001) increase in the number of POFs. It also increased the ovulation rate by 67.4% (P < 0.01), with a higher proportion (χ2df1 = 10.7, P < 0.001) of ewes in the MET group having multiple ovulations compared to the CON group (82.6 vs. 30.4%). With 1.48 ± 0.12 prolificacy rate in MET ewes, the proportion of ewes giving birth to multiple lambs was 2.9-fold higher than in the CON group. Plasma estradiol, insulin, glucose, total cholesterol, and LDL-cholesterol concentrations were lower (P < 0.05) in the MET ewes than in the CON. The results of the present study indicate that metformin can increase the number of POF, ovulation rate, fetal rate and prolificacy in ewes, while reducing the plasma estradiol, insulin, glucose and cholesterol in MET ewes.

To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary.
Descriptive study.
University Hospital.
The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving in vitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation.
None.
MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles.
Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue.
This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.

Ligusticum chuanxiong (CX) is a traditional Chinese medicine that is widely planted throughout the world. CX is one of the most important and commonly used drugs to enhance blood circulation. The preovulatory follicles in laying hens have a large number of blood arteries and meridians that feed the follicles\' growth and maturation with nutrients, hormones, and cytokines. With the extension of laying time, preovulatory follicles angiogenesis decreased gradually. In this study, we studied the mechanism of CX on preovulatory follicles angiogenesis in late-phase laying hens. The results show that CX extract can increase the angiogenesis of preovulatory follicles (F1-F3) of late-phase laying hens. CX extract can promote vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation in preovulatory follicles theca layers, promote the proliferation, invasion and migration through PI3K/AKT and RAS/ERK signaling pathways in primary follicle microvascular endothelial-like cells (FMECs). In addition, CX extract can up-regulate the expression of hypoxia inducible factor α (HIF1α) in granulosa cells (GCs) and granulosa layers through PI3K/AKT and RAS/ERK signaling pathways, thereby promoting the secretion of vascular endothelial growth factor A (VEGFA). In conclusion, the current study confirmed the promoting effect of CX extract on the preovulatory follicles angiogenesis, which sets the stage for the design of functional animal feed for late-phase laying hens.

The routine procedure of estrous cycle synchronization in pigs allows for the use of gonadotropins to stimulate ovarian activity. The applied protocols of eCG and hFSH priming similarly affected development of ovarian follicles in two classes 3−6 mm and >6 mm of diameter, however, the number of small follicles (<3 mm) was 2-fold higher in hFSH- than in eCG-primed prepubertal gilts. The attainment of sexual maturity increased concentration of estradiol, testosterone and androstenedione in the follicular fluid of hFSH/eCG-primed gilts, however, prostaglandin E2 and F2α metabolite increased in mature hFSH- and eCG-primed gilts, respectively. The maturity increased mRNA and/or protein expression of key steroidogenic enzymes, prostaglandin synthases or luteinizing hormone receptors in follicular walls. Both hormonal primers played a moderate role in affecting expression of steroidogenic enzymes in follicular walls. In vitro studies showed higher estradiol production in r-hLH (p = 0.04)- and r-hCG (p = 0.049)-stimulated follicular walls of mature gilts than in prepubertal hFSH-primed gilts. Both ovulatory triggers decreased the abundance of LHCG/FSH mRNA receptors in follicular walls, which mimic downregulation of these receptors by a preovulatory LH surge, confirmed in vivo. These data revealed the importance of sexual maturity in the protection of the estrogenic environment, and the selective, moderate role of eCG and FSH in the activation of steroidogenic enzymes in preovulatory follicles.

Ovary follicular development is a progressive system from the beginning of small cortical follicles to the ovulation of hierarchical follicles. The review was conducted to provide information on the indigenous chickens commonly used for egg production, chicken ovarian follicles morphology and expression of growth differentiation factor 9 (GDF9) gene in ovarian follicles and its relationship with egg production. The research databases used in the study include google scholar, Science Direct, PubMed, JSTOR and Cambridge Core. Google, Yahoo and Baidu search engines were used to search the information. In this study, the papers selected for use were original research articles and reviews to ensure that the information used was from research results. Besides, only recent English papers, 2010-2021, were used. The keywords used to search for articles were chicken ovarian follicles, ovarian morphology and GDF9 gene expression. The documents showed that pre-hierarchical follicles include many small and large white follicles, which are about 2-5mm in diameter and 5 to 6 small yellow follicles (SYF) that are about 5-10mm in diameter. Preovulatory follicles are about five to six in number and above 10mm in diameter, with the sizes from F6 to F1, with F1 as the largest follicle. Further, the studies revealed that GDF9 gene mRNA is expressed in the highest concentration in small yellow follicles and other studies reported that the expression of GDF9 gene has been found in follicles of the primary to preovulatory stages in chickens. This review concludes that the GDF9 gene expression is mainly throughout follicular growth and it stimulates the proliferation of pre-hierarchical granulosa cells. The increased egg production in chickens depends on progressive developmental stages and the growth of ovarian follicles.

Based on a previous global transcriptome sequencing project, we hypothesized that Lumican (LUM) might play a role in ovulatory processes. We sought to determine LUM gene expression under various conditions in human preovulatory follicles. The in vitro expression of LUM mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical staining was used to identify human LUM expression in follicles at different developmental stages. Cell signaling studies were performed by treating human MGCs with human chorionic gonadotropin (hCG) and both, different stimulators and inhibitors to determine their effect on LUM expression by using qRT-PCR. Cell confluence studies were carried out to study the correlation between LUM expression and follicle cell proliferation. Follicular MGCs and CGCs of women undergoing in vitro fertilization (IVF) procedures due to endometriosis were analyzed for differences in LUM expression patterns by qRT-PCR. LUM mRNA expression was significantly higher in MGCs as compared to CGCs. In CGCs, LUM mRNA was higher in mature metaphase II (MII) oocytes than in germinal vesicle (GV) and metaphase I (MI) oocytes. LUM expression was significantly upregulated in response to hCG in cultured MGCs. Immunohistochemistry of human ovaries revealed LUM was mostly present in MGCs of large preovulatory and postovulatory follicles and absent from primordial follicles. Using pharmacological activators and inhibitors, we demonstrated that LUM induction by luteinizing hormone (LH)/hCG is carried through the mitogen-activated protein kinase (MEK) pathway. LUM expression was induced in high-density cell cultures in a confluence-dependent manner. MGCs from follicles of subjects with endometriosis exhibited reduced mRNA transcription levels compared to control subjects. Our study confirms that LUM is a newly discovered ovulatory gene. LUM might play an important role during the preovulatory period up until ovulation as well as in endometriosis infertility. A better understanding of LUM\'s role might provide potential new treatment paradigms for some types of female infertility.

Arginine vasotocin (VT) is the basic neurohypophysial nonapeptide hormone in teleosts. VT is also distributed in the ovary of the catfish Heteropneustes fossilis and induces final oocyte maturation (FOM) and ovulation by stimulating the maturation-inducing hormone (MIH). The present study reports the effects of cAMP (0.5 mM), phosphodiesterase inhibitors (IBMX -0.5 mM and theophylline- 0.5 mM), the inositol triphosphate (IP3) receptor inhibitor heparin (10 μg/mL) and the Ca2+ chelator BAPTA-AM (25 μM) on VT (100 nM) - induced progestin stimulation, FOM and ovulation. Incubation of post-vitellogenic follicles with cAMP, IBMX and theophylline for 0, 8, 16 and 24 h stimulated basal secretion of progesterone (P4), 17-hydroxyprogesterone (17-P) and 17, 20β-dihydroxy-4-pregnen-3-one (MIH) in a duration-dependent manner. The incubation of the follicles with heparin stimulated P4 modestly, and 17-P and MIH levels in a duration-dependent manner. The incubation of the follicles with BAPTA-AM stimulated P4 and MIH levels marginally and 17-P robustly. The stimulation was in the order cAMP > IBMX > theophylline > heparin > BAPTA-AM. The incubation of the follicles with VT stimulated P4, 17-P, MIH, GVBD and ovulation in a duration-dependent manner. The co-incubations with VT and the test compounds inhibited the VT-induced stimulation of P4, 17-P and MIH levels in a time-dependent manner in the order heparin > BAPTA-AM > cAMP > IBMX > theophylline. Concurrently, the VT-induced stimulation of GVBD and ovulation were also inhibited by the test compounds in the same order. The results show that VT induces FOM and ovulation preferentially acting through Ca2+ pathway and a crosstalk between Ca2+ and cAMP signaling pathways seems to integrate the processes.

This study assessed the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the granulosa layers (GLs) and theca layers (TLs) of chicken preovulatory follicles in vitro and in vivo. In the in vitro experiment, three of the largest yellow preovulatory follicles (F3 < F2 < F1) were exposed to PNP or PNMC (10-8-10-4 M), ovine luteinising hormone (oLH; 10 ng/mL), and combinations of oLH and PNP or PNMC (10-6 M). In the in vivo experiment, laying hens were treated for 6 days with PNP or PNMC (10 mg/kg). In vitro experiments revealed that PNP and PNMC decreased basal and oLH-stimulated P4 secretion from the GL as well as T and E2 secretion from the TLs of F3-F1 follicles. Treatment of laying hens with nitrophenols lowered plasma concentrations of luteinising hormone and all three steroids. The reduction of steroid secretion was associated with decrease in LHR, HSD3B1 and CYP19A1 mRNA expression in the GL and/or TLs of the preovulatory follicles, both in vitro and in vivo. Moreover, PNP decreased HSD3B protein expression in the GL of F2 follicles in vitro and in vivo, while PNMC diminished its expression in the GL of F1 follicles in vivo. In vitro, nitrophenols did not affect CYP19A1 protein expression; however, nitrophenols inhibited its expression in the TLs of F3 and F2 follicles in vivo. The results obtained clearly demonstrate that nitrophenols are negative modulators of steroidogenesis in chicken preovulatory follicles and, in consequence, may not only impair ovulation process, but also affect function of the hypothalamic-pituitary-ovarian axis.

BACKGROUND: DCN (decorin) is a proteoglycan known to be involved in regulating cell proliferation, collagen fibril organization and migration. In our global transcriptome RNA-sequencing approach to systematically identify new ovulation-associated genes, DCN was identified as one of the highly regulated genes. We therefore hypothesize that DCN may have a role in ovulatory processes such as cell migration and proliferation.
OBJECTIVE: To characterize the expression, regulation and function of the proteoglycan DCN in the human ovarian follicles during the preovulatory period.
METHODS: The in-vivo expression of DCN mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative RT-PCR and western blot. A signaling study was performed by treating human MGCs cultures with gonadotropins and different stimulators and inhibitors to determine their effect on DCN expression by qRT- PCR and elucidate the pathways regulating these proteins. In a functional study, KGN granulosa cell line was used to study cell migration with a scratch assay.
RESULTS: DCN mRNA expression was significantly higher in MGCs compared to CGCs. DCN mRNA was significantly higher in CGCs surrounding mature metaphase II (MII) oocytes compared to CGCs of germinal vesicle (GV) and metaphase I (MI) oocytes. hCG significantly increased DCN mRNA and protein expression levels in cultured MGCs. Using signal transduction activators and inhibitors, we demonstrated that DCN induction by LH/hCG is carried out via PKA, PKC, ERK/MEK, and PI3K pathways. We showed that DCN expression is also induced in high-density cell cultures, in a dose-dependent pattern. In addition, progesterone induced a significant increase in DCN secretion to the media. MGCs from follicles of endometriosis patients exhibited reduced (about 20% of) mRNA transcriptions levels compared to MGCs follicles of control patients. More significantly, we found that DCN has an inhibiting effect on KGN cell migration.
CONCLUSIONS: Our study indicates that DCN is a unique ovulatory gene. Our findings support the hypothesis that DCN plays an important new role during the preovulatory period and ovulation, and stress its involvement in endometriosis infertility. A better understanding of DCN role in ovulation and endometriosis may provide treatment for some types of infertility.

Purpose: To illustrate whether low-dose human chorionic gonadotropin (hCG) administration during the early follicular phase could reduce the number of large preovulatory follicles in women with polycystic ovarian syndrome (PCOS) undergoing ovarian stimulation using the progesterone protocol. Methods: We performed a randomized, controlled pilot trial at a university-affiliated tertiary hospital. A total of 40 infertile women undergoing their first in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment with the freeze-all strategy were included. Human menopausal gonadotropin (hMG) and progesterone soft capsule 100 mg/d were added simultaneously beginning from menstrual cycle day 3 for all participants. Low-dose hCG (200 IU) was injected every 3 days in the study group from the first day of ovarian stimulation until trigger. The primary outcome was the number of large preovulatory follicles. Secondary outcomes included the incidence of ovarian hyperstimulation syndrome (OHSS); the number of oocytes retrieved, mature oocytes, and good-quality embryos; and clinical results after frozen-thawed embryo transfer (FET) cycles. Results: The study group had slightly more large preovulatory follicles than the control group (17.75 ± 10 vs. 13.2 ± 5.34; P > 0.05). None of the participants experienced severe OHSS. There were no statistically significant differences in the number of oocytes retrieved (15.9 ± 8.46 vs. 15.75 ± 6.96), mature oocytes (13.55 ± 6.56 vs. 13.4 ± 6.34), and good-quality embryos (5.5 ± 3.41 vs. 4.9 ± 2.99) between the two groups (P > 0.05). Clinical pregnancy rates (65.52 vs. 41.94%; P = 0.067) and live birth rates (48.28 vs. 35.48%; P = 0.315) per transfer following FET of the study group were higher than those of the control group, but without statistical significance. Conclusions: Administration of low-dose hCG from the early follicular phase for PCOS patients undergoing ovarian stimulation with progesterone protocol may lead to slightly more early preovulatory follicles and marginally, but not significantly, higher clinical pregnancy rates. A continuous trial should be performed to explore the effects of supplementation with different doses of hCG from the start of ovarian stimulation in PCOS patients using the progesterone protocol. Clinical Trial Registration: Chictr.org.cn, identifier: ChiCTR-IOR-15007165.