Relationship between LH receptor concentrations in thecal and granulosa cells and in-vivo and in-vitro steroid secretion by ovine follicles during the preovulatory period

Abstract The luteinizing hormone (LH) surge is essential for ovulation, but the intrafollicular factors induced by LH that mediate ovulatory processes (e.g., angiogenesis) are poorly understood, especially in women. The role of secretogranin II (SCG2) and its cleaved bioactive peptide, secretoneurin (SN), were investigated as potential mediators of ovulation by testing the hypothesis that SCG2/SN is induced in granulosa cells by human chorionic gonadotropin (hCG), via a downstream LH receptor signaling mechanism, and stimulates ovarian angiogenesis. Humans, nonhuman primates, and rodents were treated with hCG in vivo resulting in a significant increase in the messenger RNA and protein levels of SCG2 in granulosa cells collected early during the periovulatory period and just prior to ovulation (humans: 12 to 34 hours; monkeys: 12 to 36 hours; rodents: 4 to 12 hours post-hCG). This induction by hCG was recapitulated in an in vitro culture system utilizing granulosa-lutein cells from in vitro fertilization patients. Using this system, inhibition of downstream LH receptor signaling pathways revealed that the initial induction of SCG2 is regulated, in part, by epidermal growth factor receptor signaling. Further, human ovarian microvascular endothelial cells were treated with SN (1 to 100 ng/mL) and subjected to angiogenesis assays. SN significantly increased endothelial cell migration and new sprout formation, suggesting induction of ovarian angiogenesis. These results establish that SCG2 is increased in granulosa cells across species during the periovulatory period and that SN may mediate ovulatory angiogenesis in the human ovary. These findings provide insight into the regulation of human ovulation and fertility.

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The role of intraovarian regulators in the aetiology of polycystic ovarian syndrome

Reproductive Medicine Review ◽

10.1017/s0962279900001320 ◽

1996 ◽

Vol 5 (3) ◽

pp. 151-168 ◽

Cited By ~ 5

Author(s):

Ghanim Almahbobi ◽

Alan O Trounson

Keyword(s):

Granulosa Cells ◽

Follicular Development ◽

Aromatase Activity ◽

Lh Receptor ◽

Follicular Maturation ◽

Developmental Capacity ◽

Follicle Selection

The present review demonstrates that the availability of bioactive FSH and LH in PCOS is normal and that granulosa cells of PCO are not apoptotic and instead hyperexpress functional FSH receptors and may possess intact aromatase activity. Consequently, these cells respond excessively to exogenous FSH stimulation and produce high amounts of oestradiol both in vivo and in vitro. The altered developmental capacity of follicles from PCO in vivo is most likely due to the abnormal follicular milieu of PCO and the culminating effects of intrafollicular inhibitors and stimulators. The failure of ovarian oestradiol production and follicular maturation to dominance in vivo may be due to a mechanism that interferes with the function of FSH, such as intraovarian steroids and growth factors. It has previously been shown that EGF and TGFα have inhibitory actions on follicular development, aromatization and LH receptor formation. In contrast, EGF enhances early follicular recruitment and growth. Therefore, it is hypothesized that EGF/TGFα may have a causal relationship in the mechanisms of anovulatory infertility in women with PCOS. Thus, an aberration in the regulation of follicular fluid EGF and/or TGFα may result in reduced numbers of granulosa cells, cessation of follicle selection and ultimately in the creation and maintenance of PCOS. The exact mechanism by which the hyperfunction of EGF/TGFα occurs and the trigger for this hyperactivity in the ovary remain to be determined. An experimental animal model may be required to assist such investigations in the future.

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Thyroid hormone as a regulator of basal and human chorionic gonadotrophin-stimulated steroidogenesis by cultured porcine theca and granulosa cells isolated at different stages of the follicular phase

Reproduction Fertility and Development ◽

10.1071/rd9960961 ◽

1996 ◽

Vol 8 (6) ◽

pp. 961 ◽

Cited By ~ 33

Author(s):

EL Gregoraszczuk ◽

M Skalka

Keyword(s):

Thyroid Hormone ◽

Granulosa Cells ◽

Chorionic Gonadotrophin ◽

Human Chorionic Gonadotrophin ◽

Follicular Cells ◽

Steroid Secretion ◽

Follicle Size ◽

Androgen Secretion

To characterize thyroid hormone action in the ovary, the direct effect of triiodothyronine (T3) was investigated in vitro using a culture system of porcine theca cells (Tcs) and granulosa cells (Gcs) in mono- and co-culture (GT), the latter resembling follicles in vivo. The cells were cultured in the absence or presence of human chorionic gonadotrophin (hCG) with or without T3 (10(-7), 10(-9) or 10(-11) M). Follicular cells were obtained from follicles of different size (small, medium and large), and steroid secretion into the culture medium was detected by radioimmunoassay. T3 alone did not influence steroid secretion by Tcs and Gcs isolated from follicles that were small and medium in size. In preovulatory follicles, an increase in basal androgen secretion and a simultaneous decrease in oestradiol secretion were observed with Tcs and Gcs in both mono- and co-culture. T3 together with hCG decreased hCG-stimulated androgen secretion in Tcs isolated from medium-sized follicles and had a simultaneous stimulatory effect on hCG-stimulated oestradiol secretion by Gcs. In cultures of follicular cells obtained from large follicles, T3 decreased hCG-stimulated secretion of both androgen and oestrogen by Tcs and simultaneously stimulated oestradiol secretion in GT co-cultures. Thus, the interaction of T3 with gonadotrophin hormone modulated follicular steroidogenesis, depending on follicle size and cell type used in culture. The observed T3-induced increase in basal androgen secretion by Tcs could account for the atresia of follicles, since it is accompanied by a decrease in oestradiol secretion in GT co-culture. In its co-activity with hCG, an adequate level of T3 prevents excessive androgen production by Tcs, probably influencing aromatization processes in the follicle. An increase in hCG-stimulated oestradiol secretion in GT co-cultures is then observed. Further investigations are required to clarify whether this is linked with an effect on the aromatization processes occurring in the follicle.

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The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

Reproduction ◽

10.1530/rep-09-0518 ◽

2010 ◽

Vol 139 (4) ◽

pp. 759-769 ◽

Cited By ~ 42

Author(s):

F P Yuan ◽

X Li ◽

J Lin ◽

C Schwabe ◽

E E Büllesbach ◽

...

Keyword(s):

Mesenchymal Cell ◽

Postnatal Period ◽

Testosterone Replacement Therapy ◽

Developmental Defect ◽

Mrna Levels ◽

Lh Receptor ◽

Protein Levels ◽

Testis Descent

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT).In vivoandin vitroexperiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development inLhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression ofRxfp2which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase inRxfp2mRNA levels in a time-dependent fashion inLhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediatedRxfp2knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent inLhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

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MiRNA-143 mediates the proliferative signaling pathway of FSH and regulates estradiol production

Journal of Endocrinology ◽

10.1530/joe-16-0488 ◽

2017 ◽

Vol 234 (1) ◽

pp. 1-14 ◽

Cited By ~ 19

Author(s):

Li Zhang ◽

XiaoXin Zhang ◽

Xuejing Zhang ◽

Yu Lu ◽

Lei Li ◽

...

Keyword(s):

Signaling Pathway ◽

Granulosa Cell ◽

Granulosa Cells ◽

Loss Of Function ◽

Cellular Processes ◽

Cell Functions ◽

Genes Expression ◽

Underlying Mechanisms

MicroRNAs (MiRNAs) play important regulatory roles in many cellular processes. MiR-143 is highly enriched in the mouse ovary, but its roles and underlying mechanisms are not well understood. In the current study, we show that miR-143 is located in granulosa cells of primary, secondary and antral follicles. To explore the specific functions of miR-143, we transfected miR-143 inhibitor into primary cultured granulosa cells to study the loss of function of miR-143 and the results showed that miR-143 silencing significantly increased estradiol production and steroidogenesis-related gene expression. Moreover, our in vivo and in vitro studies showed that follicular stimulating hormone (FSH) significantly decreased miR-143 expression. This function of miR-143 is accomplished by its binding to the 3’-UTR of KRAS mRNA. Furthermore, our results demonstrated that miR-143 acts as a negative regulating molecule mediating the signaling pathway of FSH and affecting estradiol production by targeting KRAS. MiR-143 also negatively acts in regulating granulosa cells proliferation and cell cycle-related genes expression. These findings indicate that miR-143 plays vital roles in FSH-induced estradiol production and granulosa cell proliferation, providing a novel mechanism that involves miRNA in regulating granulosa cell functions.

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Investigating the Intrafollicular Factors Affecting Oocyte Developmental Competency

10.26686/wgtn.17068070.v1 ◽

2021 ◽

Author(s):

◽

Zaramasina Clark

Keyword(s):

Gene Expression ◽

Oocyte Maturation ◽

Granulosa Cells ◽

Embryo Quality ◽

Cumulus Cells ◽

Significant Finding ◽

High Quality ◽

Final Maturation

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos. The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes. The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation. The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>

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Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Reproduction ◽

10.1530/rep-12-0228 ◽

2013 ◽

Vol 145 (2) ◽

pp. 127-135 ◽

Cited By ~ 6

Author(s):

Nazareth Loreti ◽

Verónica Ambao ◽

Luz Andreone ◽

Romina Trigo ◽

Ursula Bussmann ◽

...

Keyword(s):

Granulosa Cells ◽

Concanavalin A ◽

Inhibin B ◽

Inhibin A ◽

Biological Responses ◽

Immature Rats ◽

Fold Stimulation ◽

Recombinant Human Fsh

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

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