Role of leptin receptors in granulosa cells during ovulation

Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 μg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

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6 CRISPR-Generated ZP4 Knockout Rabbits Exhibit Impaired In Vivo Pre-Implantation Development

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab6 ◽

2018 ◽

Vol 30 (1) ◽

pp. 142

Author(s):

I. Lamas-Toranzo ◽

N. F. Balvís ◽

P. L. Lorenzo ◽

P. G. Rebollar ◽

M. Avilés ◽

...

Keyword(s):

Embryo Development ◽

Zona Pellucida ◽

Critical Role ◽

Laboratory Mouse ◽

Follicular Development ◽

Reproductive Function ◽

Small Litter ◽

Morphological Differences

Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P < 0.05). These morphological differences suggest that rabbit ZP without ZP4 may fail to protect the embryo during development or may physically impede blastocyst expansion. This study is supported by the projects AGL2014-58739-R and RYC-2012-10193 (to PBA), AGL2015-70159-P (to MA) and AGL2015-65572-C2-1-R (to PGR and PL). ILT and NFB are supported by FPI grants.

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MicroRNA Mediating Networks in Granulosa Cells Associated with Ovarian Follicular Development

BioMed Research International ◽

10.1155/2017/4585213 ◽

2017 ◽

Vol 2017 ◽

pp. 1-18 ◽

Cited By ~ 4

Author(s):

Baoyun Zhang ◽

Long Chen ◽

Guangde Feng ◽

Wei Xiang ◽

Ke Zhang ◽

...

Keyword(s):

Transcription Factors ◽

Granulosa Cells ◽

Messenger Rna ◽

Expression Patterns ◽

Follicular Development ◽

Functional Networks ◽

Signal Pathways ◽

Differentially Expressed Mirnas ◽

Selection Of

Ovaries, which provide a place for follicular development and oocyte maturation, are important organs in female mammals. Follicular development is complicated physiological progress mediated by various regulatory factors including microRNAs (miRNAs). To demonstrate the role of miRNAs in follicular development, this study analyzed the expression patterns of miRNAs in granulosa cells through investigating three previous datasets generated by Illumina miRNA deep sequencing. Furthermore, via bioinformatic analyses, we dissected the associated functional networks of the observed significant miRNAs, in terms of interacting with signal pathways and transcription factors. During the growth and selection of dominant follicles, 15 dysregulated miRNAs and 139 associated pathways were screened out. In comparison of different styles of follicles, 7 commonly abundant miRNAs and 195 pathways, as well as 10 differentially expressed miRNAs and 117 pathways in dominant follicles in comparison with subordinate follicles, were collected. Furthermore, SMAD2 was identified as a hub factor in regulating follicular development. The regulation of miR-26a/b onsmad2messenger RNA has been further testified by real time PCR. In conclusion, we established functional networks which play critical roles in follicular development including pivotal miRNAs, pathways, and transcription factors, which contributed to the further investigation about miRNAs associated with mammalian follicular development.

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Laminin-alpha6beta1 integrin interaction enhances survival and proliferation and modulates steroidogenesis of ovine granulosa cells

Journal of Endocrinology ◽

10.1677/joe.0.1720045 ◽

2002 ◽

Vol 172 (1) ◽

pp. 45-59 ◽

Cited By ~ 30

Author(s):

F Le Bellego ◽

C Pisselet ◽

C Huet ◽

P Monget ◽

D Monniaux

Keyword(s):

Estrous Cycle ◽

Granulosa Cells ◽

Physiological Role ◽

Follicular Development ◽

Antral Follicles ◽

Final Development ◽

Beta 1 Integrin ◽

Arginine Glycine Aspartic Acid

This study aimed to determine the physiological role of laminin (LN) and its receptor, alpha(6)beta(1) integrin, in controlling the functions of granulosa cells (GC) during follicular development in sheep ovary. Immunohistochemistry experiments showed the presence of increasing levels of LN (P<0.0001), and high levels of mature alpha(6)beta(1) integrin in GC layers of healthy antral follicles during the follicular and the preovulatory phases of the estrous cycle. In vitro, the addition of a function-blocking antibody raised against alpha(6) subunit (anti-alpha(6) IgG) to the medium of ovine GC cultured on LN impaired cell spreading (P<0.0001), decreased the proliferation rate (P<0.05) and increased the apoptosis rate (P<0.05). Furthermore, addition of anti-alpha(6) IgG enhanced estradiol (E2) secretion by GC in the presence or absence of follicle-stimulating hormone (FSH), luteinizing hormone or insulin-like growth factor-I in culture medium (P<0.0001), and inhibited progesterone (P4) secretion in basal conditions or in the presence of low (0.5 ng/ml) FSH concentrations only (P<0.0001). The anti-alpha(6) IgG effect was specific to an interaction of LN with alpha(6)beta(1) integrin since it was ineffective on GC cultured on heat-denatured LN, RGD (arginine-glycine-aspartic acid) peptides and non-coated substratum. Hence, this study established that alpha(6)beta(1) integrin 1) was expressed in GC of antral follicles, 2) mediated the actions of LN on survival, proliferation and steroidogenesis of GC, and 3) was able to dramatically modulate P4 and E2 secretion by GC in vitro. It is suggested that during the follicular and the preovulatory phases of the estrous cycle, the increasing levels of LN in GC of large antral follicles might support their final development to ovulation.

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Female Fertility Is Reduced in Mice Lacking Ca2+/Calmodulin-Dependent Protein Kinase IV**This work was supported by an NIH Medical Scientist Training Program award (to J.Y.W.) and NIH Grants HD-07503 (to A.R.M.) and HD-1272 (to J.S.R.)

Endocrinology ◽

10.1210/endo.141.12.7826 ◽

2000 ◽

Vol 141 (12) ◽

pp. 4777-4783 ◽

Cited By ~ 27

Author(s):

Joy Y. Wu ◽

Ignacio J. Gonzalez-Robayna ◽

JoAnne S. Richards ◽

Anthony R. Means

Keyword(s):

Protein Kinase ◽

Granulosa Cells ◽

Critical Role ◽

Follicular Development ◽

Female Fertility ◽

Female Reproduction ◽

Dependent Protein Kinase ◽

Medical Scientist ◽

Dependent Protein ◽

Medical Scientist Training Program

Abstract Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.

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Variations in Adipokine GenesAdipoQ,Lep, andLepRAre Associated with Risk for Obesity-Related Metabolic Disease: The Modulatory Role of Gene-Nutrient Interactions

Journal of Obesity ◽

10.1155/2011/168659 ◽

2011 ◽

Vol 2011 ◽

pp. 1-17 ◽

Cited By ~ 31

Author(s):

Jennifer Emily Enns ◽

Carla G. Taylor ◽

Peter Zahradka

Keyword(s):

Metabolic Disease ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Disease Risk ◽

Critical Role ◽

Nucleotide Polymorphisms ◽

Nutrient Interactions ◽

The Metabolic Syndrome ◽

Susceptible Populations

Obesity rates are rapidly increasing worldwide and facilitate the development of many related disease states, such as cardiovascular disease, the metabolic syndrome, type 2 diabetes mellitus, and various types of cancer. Variation in metabolically important genes can have a great impact on a population's susceptibility to becoming obese and/or developing related complications. The adipokines adiponectin and leptin, as well as the leptin receptor, are major players in the regulation of body energy homeostasis and fat storage. This paper summarizes the findings of single nucleotide polymorphisms in these three genes and their effect on obesity and metabolic disease risk. Additionally, studies of gene-nutrient interactions involving adiponectin, leptin, and the leptin receptor are highlighted to emphasize the critical role of diet in susceptible populations.

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Gene analysis of major signaling pathways regulated by gonadotropins in human ovarian granulosa tumor cells (KGN)†

Biology of Reproduction ◽

10.1093/biolre/ioaa079 ◽

2020 ◽

Vol 103 (3) ◽

pp. 583-598

Author(s):

Patricia G Tremblay ◽

Marc-André Sirard

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Signaling Pathways ◽

Tumor Cells ◽

Granulosa Cell ◽

Granulosa Cells ◽

Follicular Development ◽

Reproductive Function ◽

New Information ◽

Pkc Signaling

Abstract The female reproductive function largely depends on timing and coordination between follicle-stimulating hormone (FSH) and luteinizing hormone. Even though it was suggested that these hormones act on granulosa cells via shared signaling pathways, mainly protein kinases A, B, and C (PKA, PKB, and PKC), there is still very little information available on how these signaling pathways are regulated by each hormone to provide such differences in gene expression throughout folliculogenesis. To obtain a global picture of the principal upstream factors involved in PKA, PKB, and PKC signaling in granulosa cells, human granulosa-like tumor cells (KGN) were treated with FSH or specific activators (forskolin, SC79, and phorbol 12-myristate 13-acetate) for each pathway to analyze gene expression with RNA-seq technology. Normalization and cutoffs (FC 1.5, P ≤ 0.05) revealed 3864 differentially expressed genes between treatments. Analysis of major upstream regulators showed that PKA is a master kinase of early cell differentiation as its activation resulted in the gene expression profile that accompanies granulosa cell differentiation. Our data also revealed that the activation of PKC in granulosa cells is also a strong differentiation signal that could control “advanced” differentiation in granulosa cells and the inflammatory cascade that occurs in the dominant follicle. According to our results, PKB activation provides support for PKA-stimulated gene expression and is also involved in granulosa cell survival throughout follicular development. Taken together, our results provide new information on PKA, PKB, and PKC signaling pathways and their roles in stimulating a follicle at the crossroad between maturation/ovulation and atresia.

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Expression of Functional Leptin Receptors in the Human Ovary1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.82.12.4446 ◽

1997 ◽

Vol 82 (12) ◽

pp. 4144-4148 ◽

Cited By ~ 5

Author(s):

Cecilia Karlsson ◽

Kajsa Lindell ◽

Eva Svensson ◽

Christina Bergh ◽

Peter Lind ◽

...

Keyword(s):

Adipose Tissue ◽

Granulosa Cells ◽

Follicular Fluid ◽

Leptin Receptor ◽

Biological Response ◽

Human Ovary ◽

Leptin Receptors ◽

Short Isoforms ◽

Reverse Transcription Pcr ◽

Ovarian Cells

The size of body fat stores is known to influence fertility, indicating a link between adipose tissue and the reproductive system. Studies in mice have identified the adipocyte-derived hormone, leptin (Ob protein), as a possible mediator of this effect. The aim of this study was to investigate the possibility that leptin may have direct effects on the human ovary. To probe this hypothesis we first analyzed the expression of leptin receptors in the human ovary. Transcripts encoding both the long and short isoforms of the leptin receptor were present in human granulosa cells and thecal cells; however, the short isoforms were expressed at much higher levels. Immunoreactive leptin was present in follicular fluid at levels similar to those found in serum. ob gene expression, however, was undetectable in the ovary, as determined by reverse transcription-PCR, whereas it was easily detected in adipose tissue. To determine whether leptin could induce a biological response in ovarian cells, we examined the effect of leptin on estradiol production in cultured granulosa cells. Leptin (100 ng/mL) inhibited LH (0.1 ng/mL)-stimulated estradiol production. In contrast, leptin had no effect on estradiol production in the absence of LH. In conclusion, this study has demonstrated that the leptin receptor is expressed in the human ovary, that leptin is present in follicular fluid, and that leptin can induce a biological response in ovarian cells. These results suggest that leptin may have a direct effect on the human ovary.

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Relative Importance of the Arcuate and Anteroventral Periventricular Kisspeptin Neurons in Control of Puberty and Reproductive Function in Female Rats

Endocrinology ◽

10.1210/en.2014-1655 ◽

2015 ◽

Vol 156 (7) ◽

pp. 2619-2631 ◽

Cited By ~ 29

Author(s):

M. H. Hu ◽

X. F. Li ◽

B. McCausland ◽

S. Y. Li ◽

R. Gresham ◽

...

Keyword(s):

Pubertal Timing ◽

Critical Role ◽

Reproductive Function ◽

Pulse Frequency ◽

Pulse Generation ◽

Female Rats ◽

Relative Importance ◽

Lh Surge ◽

Estrous Cyclicity

Kisspeptin plays a critical role in pubertal timing and reproductive function. In rodents, kisspeptin perikarya within the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei are thought to be involved in LH pulse and surge generation, respectively. Using bilateral microinjections of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC or AVPV of female rats at postnatal day 10, we investigated the relative importance of these two kisspeptin populations in the control of pubertal timing, estrous cyclicity, and LH surge and pulse generation. A 37% knockdown of kisspeptin in the AVPV resulted in a significant delay in vaginal opening and first vaginal estrous, abnormal estrous cyclicity, and reduction in the occurrence of spontaneous LH surges, although these retained normal amplitude. This AVPV knockdown had no effect on LH pulse frequency, measured after ovariectomy. A 32% reduction of kisspeptin in the ARC had no effect on the onset of puberty but resulted in abnormal estrous cyclicity and decreased LH pulse frequency. Additionally, the knockdown of kisspeptin in the ARC decreased the amplitude but not the incidence of LH surges. These results might suggest that the role of AVPV kisspeptin in the control of pubertal timing is particularly sensitive to perturbation. In accordance with our previous studies, ARC kisspeptin signaling was critical for normal pulsatile LH secretion in female rats. Despite the widely reported role of AVPV kisspeptin neurons in LH surge generation, this study suggests that both AVPV and ARC populations are essential for normal LH surges and estrous cyclicity.

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Physiological and Pathological Androgen Actions in the Ovary

Endocrinology ◽

10.1210/en.2019-00101 ◽

2019 ◽

Vol 160 (5) ◽

pp. 1166-1174 ◽

Cited By ~ 11

Author(s):

Olga Astapova ◽

Briaunna M N Minor ◽

Stephen R Hammes

Keyword(s):

Granulosa Cells ◽

Polycystic Ovary ◽

Follicular Development ◽

Follicle Development ◽

Female Reproduction ◽

Negative Effects ◽

Androgen Excess ◽

Male Sex ◽

Androgen Effects

Abstract Androgens, although traditionally thought to be male sex steroids, play important roles in female reproduction, both in healthy and pathological states. This mini-review focuses on recent advances in our knowledge of the role of androgens in the ovary. Androgen receptor (AR) is expressed in oocytes, granulosa cells, and theca cells, and is temporally regulated during follicular development. Mouse knockout studies have shown that AR expression in granulosa cells is critical for normal follicular development and subsequent ovulation. In addition, androgens are involved in regulating dynamic changes in ovarian steroidogenesis that are critical for normal cycling. Androgen effects on follicle development have been incorporated into clinical practice in women with diminished ovarian reserve, albeit with limited success in available literature. At the other extreme, androgen excess leads to disordered follicle development and anovulatory infertility known as polycystic ovary syndrome (PCOS), with studies suggesting that theca cell AR may mediate many of these negative effects. Finally, both prenatal and postnatal animal models of androgen excess have been developed and are being used to study the pathophysiology of PCOS both within the ovary and with regard to overall metabolic health. Taken together, current scientific consensus is that a careful balance of androgen activity in the ovary is necessary for reproductive health in women.

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CBX2 and the Ovary: Novel Insights into Regulatory Networks in Humans

Journal of Gynecology & Reproductive Medicine ◽

10.33140/jgrm/02/01/00002 ◽

2018 ◽

Vol 2 (1) ◽

Cited By ~ 1

Keyword(s):

Ovarian Cancer ◽

Granulosa Cells ◽

Regulatory Networks ◽

Polycystic Ovary ◽

Follicular Development ◽

Female Gonad ◽

Ovary Syndrome ◽

Interacting Protein ◽

Human Ovaries

Background: Chromobox protein homolog 2 (CBX2) is a DNA-interacting protein present in humans in two isoforms, CBX2.1 and CBX2.2. Isoform-1 promotes testis development, butthe role of both variants in the female gonad pathway remains unknown. Methods: To understand its function in human ovaries, we evaluated expression of known female genes i.e.FOXL2, RSPO1 and WNT4 in human pre-granulosa cells after forced expression and RNA interference of CBX2 isoforms and vice versa. Results: The two isoforms appeared to be functionally distinct. CBX2.1 has proved to be a pro-male by enhancing SOX9, SF1 and androgen receptor (AR) expression, whereas CBX2.2 might be partly pro-female most likely via interaction with RSPO1 and the regulation of ovary developmental markers (i.e. OCT-4, AMH and ERβ). Conclusions: Both CBX2 isoforms might act as distinct regulatory agents protecting granulosa cells from uncontrolled growth and proliferation by adjusting WNT4 and RSPO1 signaling pathways, and participate to follicular development and fate by regulating the expression of SF1 and AR, thus potentially influencing fertility, menopause, ovarian cancer and perhaps polycystic ovary syndrome.

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