Skip to main content
SpringerLink
Log in

Effect of luteinizing hormone concentration on transcriptome and subcellular organelle phenotype of ovarian granulosa cells

  • Reproductive Physiology and Disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Research question

Granulosa cells (GCs) surrounding oocytes are crucial for follicular growth, oocyte development, ovulation, and luteinization under the dynamic co-stimulation of follicle stimulating hormone (FSH) and luteinizing hormone (LH). This study aimed to investigate the effect of LH levels on GCs in preovulatory follicles under gonadotropin releasing hormone antagonist-based ovarian stimulation. In vitro experiments were also conducted to study the direct effect of LH on GCs.

Methods

Twelve infertile women were divided into low (L), medium (M), and high (H) LH groups according to their serum LH levels during ovarian stimulation. RNA-sequencing (RNA-seq) was conducted to examine the transcriptome profiles of GCs obtained from the above patients during the oocyte retrieval. The activity of mitochondrial dehydrogenase was measured under the stimulation of recombinant LH (rLH) concentration gradient combined with recombinant FSH. The ultrastructures of subcellular organelles were observed.

Results

Bioinformatic analyses showed that compared with the M group, molecule and pathway changes in the L group and in the H group were similar. In cultured GCs, both insufficient and excessive rLH impaired the activity of mitochondrial dehydrogenase. With the medium rLH concentration, numerous cell connections and abundant mitochondria and liposomes were observed. Compared with the medium concentration, GCs showed smaller and rounder mitochondria, more autophagosomes, and massive organelles damages with excessive rLH, and swollen, circular, or forked mitochondria were observed with inadequate rLH.

Conclusions

RNA-seq provided a novel spectrum of transcriptome characteristics of GCs potentially affected by serum LH levels during ovarian stimulation. In vitro, rLH could directly affect GCs at the subcellular level.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Fontana J, Martínková S, Petr J, Žalmanová T, Trnka J. Metabolic cooperation in the ovarian follicle. Physiol Res. 2020;69:33–48.

    Article  CAS  PubMed  Google Scholar 

  2. Cecconi S, Ciccarelli C, Barberi M, Macchiarelli G, Canipari R. Granulosa cell-oocyte interactions. Eur J Obstet Gynecol Reprod Biol. 2004;115(Suppl 1):S19–22.

    Article  PubMed  Google Scholar 

  3. Fleming R, Jenkins J. The source and implications of progesterone rise during the follicular phase of assisted reproduction cycles. Reprod BioMed Online. 2010;21:446–9.

    Article  CAS  PubMed  Google Scholar 

  4. Jeppesen JV, Kristensen SG, Nielsen ME, Humaidan P, Dal Canto M, Fadini R, et al. LH-receptor gene expression in human granulosa and cumulus cells from antral and preovulatory follicles. J Clin Endocrinol Metab. 2012;97:E1524–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Erickson GF, Wang C, Hsueh AJ. FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature England. 1979;279:336–8.

    Article  CAS  Google Scholar 

  6. Chen L, Wert SE, Hendrix EM, Russell PT, Cannon M, Larsen WJ. Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass. Mol Reprod Dev. 1990;26:236–47.

    Article  PubMed  Google Scholar 

  7. Thanaboonyawat I, Makemaharn O, Petyim S, Laokirkkiat P, Choavaratana R. The correlation of cumulus mucification patterns with oocyte maturation rate in vitro in FSH + LH-primed IVM cycles: a prospective study. Arch Gynecol Obstet. 2016;293:681–6.

    Article  PubMed  Google Scholar 

  8. Yang S-H, Son W-Y, Yoon S-H, Ko Y, Lim J-H. Correlation between in vitro maturation and expression of LH receptor in cumulus cells of the oocytes collected from PCOS patients in HCG-primed IVM cycles. Hum Reprod. 2005;20:2097–103.

    Article  CAS  PubMed  Google Scholar 

  9. Salustri A. Paracrine actions of oocytes in the mouse pre-ovulatory follicle. Int J Dev Biol. 2000;44:591–7.

    CAS  PubMed  Google Scholar 

  10. Zhang D, Xia L, Xu H, Chen Q, Jin B, Zhang A, et al. Flexible low-dose GnRH antagonist protocol is effective in patients with sufficient ovarian reserve in IVF. Front Endocrinol (Lausanne). 2018;9:767.

    Article  Google Scholar 

  11. Liu M, Liu S, Li L, Wang P, Li H, Li Y. LH levels may be used as an indicator for the time of antagonist administration in GnRH antagonist protocols-a proof-of-concept study. Front Endocrinol (Lausanne). 2019;10:67.

    Article  Google Scholar 

  12. Lisi F. To add or not to add LH: comments on recent commentaries. Reprod BioMed Online. 2006;12:415–7.

    Article  CAS  PubMed  Google Scholar 

  13. Recombinant human luteinizing hormone (LH) to support recombinant human follicle-stimulating hormone (FSH)-induced follicular development in LH- and FSH-deficient anovulatory women: a dose-finding study. The European Recombinant Human LH Study Group. J Clin Endocrinol Metab. 1998;83:1507–14.

  14. Hillier SG. Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis. Hum Reprod. 1994;9:188–91.

    Article  CAS  PubMed  Google Scholar 

  15. Häggström M. Reference ranges for estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone during the menstrual cycle. Wikiversity J Med 2014;1.

  16. Stricker R, Eberhart R, Chevailler M-C, Quinn FA, Bischof P, Stricker R. Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during different phases of the menstrual cycle on the Abbott ARCHITECT analyzer. Clin Chem Lab Med. 2006;44:883–7.

    Article  CAS  PubMed  Google Scholar 

  17. Trenkić M, Popović J, Kopitović V, Bjelica A, Živadinović R, Pop-Trajković S. Flexible GnRH antagonist protocol vs. long GnRH agonist protocol in patients with polycystic ovary syndrome treated for IVF: comparison of clinical outcome and embryo quality. Ginekol Pol. 2016;87:265–70.

    Article  PubMed  Google Scholar 

  18. Regan L, Owen EJ, Jacobs HS. Hypersecretion of luteinising hormone, infertility, and miscarriage. Lancet. 1990;336:1141–4.

    Article  CAS  PubMed  Google Scholar 

  19. Streda R, Mardesic T, Sobotka V, Tosner J. Long GnRH agonist vs. GnRH antagonist protocol in randomized controlled trial in unselected patients--hormonal and cycle characteristics--pilot study. Ceska Gynekol. 2009;74:75–80.

    CAS  PubMed  Google Scholar 

  20. Li R, Li Y, Kristiansen K, Wang J. SOAP: short oligonucleotide alignment program. Bioinformatics. 2008;24:713–4.

    Article  CAS  PubMed  Google Scholar 

  21. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12:357–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012;9:357–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Abdi H. The Bonferonni and Šidák corrections for multiple comparisons. Encyclopedia of Measurement and Statistics 2007;3.

  26. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Su G, Morris JH, Demchak B, Bader GD. Biological network exploration with Cytoscape 3. Curr Protoc Bioinformatics. 2014;47:8.13.1–24.

    Article  Google Scholar 

  28. Casarini L, Riccetti L, De Pascali F, Nicoli A, Tagliavini S, Trenti T, et al. Follicle-stimulating hormone potentiates the steroidogenic activity of chorionic gonadotropin and the anti-apoptotic activity of luteinizing hormone in human granulosa-lutein cells in vitro. Mol Cell Endocrinol. 2016;422:103–14.

    Article  CAS  PubMed  Google Scholar 

  29. Nordhoff V, Sonntag B, von Tils D, Götte M, Schüring AN, Gromoll J, et al. Effects of the FSH receptor gene polymorphism p.N680S on cAMP and steroid production in cultured primary human granulosa cells. Reprod Biomed Online. 2011;23:196–203.

    Article  CAS  PubMed  Google Scholar 

  30. Kol S. Individualized treatment from theory to practice: the private case of adding LH during GnRH antagonist-based stimulation protocol. Clin Med Insights Reprod Health. 2014;8:59–64.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Alviggi C, Pettersson K, Longobardi S, Andersen CY, Conforti A, De Rosa P, et al. A common polymorphic allele of the LH beta-subunit gene is associated with higher exogenous FSH consumption during controlled ovarian stimulation for assisted reproductive technology. Reprod Biol Endocrinol. 2013;11:51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Shoham Z. The clinical therapeutic window for luteinizing hormone in controlled ovarian stimulation. Fertil Steril. 2002;77:1170–7.

    Article  PubMed  Google Scholar 

  33. Duffy DM. Novel contraceptive targets to inhibit ovulation: the prostaglandin E2 pathway. Hum Reprod Update. 2015;21:652–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Poulsen L la C, Englund ALM, Wissing MLM, Yding Andersen C, Borup R, Grøndahl ML. Human granulosa cells function as innate immune cells executing an inflammatory reaction during ovulation: a microarray analysis. Mol Cell Endocrinol. 2019;486:34–46.

    Article  Google Scholar 

  35. Price JC, Sheldon IM. Granulosa cells from emerged antral follicles of the bovine ovary initiate inflammation in response to bacterial pathogen-associated molecular patterns via Toll-like receptor pathways. Biol Reprod. 2013;89:119.

    Article  PubMed  Google Scholar 

  36. Shimada M, Hernandez-Gonzalez I, Gonzalez-Robanya I, Richards JS. Induced expression of pattern recognition receptors in cumulus oocyte complexes: novel evidence for innate immune-like functions during ovulation. Mol Endocrinol. 2006;20:3228–39.

    Article  CAS  PubMed  Google Scholar 

  37. Espey LL. Ovulation as an inflammatory reaction--a hypothesis. Biol Reprod. 1980;22:73–106.

    Article  CAS  PubMed  Google Scholar 

  38. Espey LL. Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod. 1994;50:233–8.

    Article  CAS  PubMed  Google Scholar 

  39. LeMaire GS. The luteinized unruptured follicle syndrome: anovulation in disguise. J Obstet Gynecol Neonatal Nurs. 1987;16:116–20.

    Article  CAS  PubMed  Google Scholar 

  40. Bashir ST, Baerwald AR, Gastal MO, Pierson RA, Gastal EL. Follicle growth and endocrine dynamics in women with spontaneous luteinized unruptured follicles versus ovulation. Hum Reprod. 2018;33:1130–40.

    Article  CAS  PubMed  Google Scholar 

  41. Tomioka RB, Ferreira GRV, Aikawa NE, Maciel GAR, Serafini PC, Sallum AM, et al. Non-steroidal anti-inflammatory drug induces luteinized unruptured follicle syndrome in young female juvenile idiopathic arthritis patients. Clin Rheumatol. 2018;37:2869–73.

    Article  PubMed  Google Scholar 

  42. Esparza LA, Schafer D, Ho BS, Thackray VG, Kauffman AS. Hyperactive LH pulses and elevated kisspeptin and NKB gene expression in the arcuate nucleus of a PCOS mouse model. Endocrinology. 2020;161.

  43. Pierre A, Peigné M, Grynberg M, Arouche N, Taieb J, Hesters L, et al. Loss of LH-induced down-regulation of anti-Müllerian hormone receptor expression may contribute to anovulation in women with polycystic ovary syndrome. Hum Reprod. 2013;28:762–9.

    Article  CAS  PubMed  Google Scholar 

  44. Yuan P, He Z, Zheng L, Wang W, Li Y, Zhao H, et al. Genetic evidence of “genuine” empty follicle syndrome: a novel effective mutation in the LHCGR gene and review of the literature. Hum Reprod. 2017;32:944–53.

    Article  CAS  PubMed  Google Scholar 

  45. Lok F, Pritchard J, Lashen H. Successful treatment of empty follicle syndrome by triggering endogenous LH surge using GnRH agonist in an antagonist down-regulated IVF cycle. Hum Reprod. 2003;18:2079–81.

    Article  CAS  PubMed  Google Scholar 

  46. Hassani F. Downregulation of extracellular matrix and cell adhesion molecules in cumulus cells of infertile polycystic ovary syndrome women with and without insulin resistance. Cell J. 2019;21:8.

    Google Scholar 

  47. Ohira T, Murayama C, Shimizu T, Yoshimura Y, Isobe N. Comparison of cadherin and integrin localization in bovine cystic and healthy follicles. Anim Sci J. 2013;84:303–9.

    Article  CAS  PubMed  Google Scholar 

  48. Thibault C, Levasseur MC. Ovulation. Hum Reprod. 1988;3:513–23.

    Article  CAS  PubMed  Google Scholar 

  49. Miner JH, Li C, Mudd JL, Go G, Sutherland AE. Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation. Development. 2004;131:2247–56.

    Article  CAS  PubMed  Google Scholar 

  50. Hu L, Zang M, Wang H-X, Li J-F, Su L-P, Yan M, et al. Biglycan stimulates VEGF expression in endothelial cells by activating the TLR signaling pathway. Mol Oncol. 2016;10:1473–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Imai K, Khandoker MAMY, Yonai M, Takahashi T, Sato T, Ito A, et al. Matrix metalloproteinases-2 and -9 activities in bovine follicular fluid of different-sized follicles: relationship to intra-follicular inhibin and steroid concentrations. Domest Anim Endocrinol. 2003;24:171–83.

    Article  CAS  PubMed  Google Scholar 

  52. Pei M, Luo J, Chen Q. Enhancing and maintaining chondrogenesis of synovial fibroblasts by cartilage extracellular matrix protein matrilins. Osteoarthr Cartil. 2008;16:1110–7.

    Article  CAS  Google Scholar 

  53. Ishikawa T, Kramer RH. Sdc1 negatively modulates carcinoma cell motility and invasion. Exp Cell Res. 2010;316:951–65.

    Article  CAS  PubMed  Google Scholar 

  54. Zhu T, Zhang X. Research progress on the role of epithelial-mesenchymal transition in pathogenesis of endometriosis. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2016;45:439–45.

    PubMed  Google Scholar 

  55. Proestling K, Birner P, Gamperl S, Nirtl N, Marton E, Yerlikaya G, et al. Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis. Reprod Biol Endocrinol. 2015;13:75.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Yang Y-M, Yang W-X. Epithelial-to-mesenchymal transition in the development of endometriosis. Oncotarget. 2017;8:41679–89.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Feng D, Zhao T, Yan K, Liang H, Liang J, Zhou Y, et al. Gonadotropins promote human ovarian cancer cell migration and invasion via a cyclooxygenase 2-dependent pathway. Oncol Rep. 2017;38:1091–8.

    Article  CAS  PubMed  Google Scholar 

  58. Catt KJ, Dufau ML. Peptide hormone receptors. Annu Rev Physiol. 1977;39:529–57.

    Article  CAS  PubMed  Google Scholar 

  59. Fraser HM, Tsonis CG. Manipulation of inhibin during the luteal-follicular phase transition of the primate menstrual cycle fails to affect FSH secretion. J Endocrinol. 1994;142:181–6.

    Article  CAS  PubMed  Google Scholar 

  60. Gomes LC, Di Benedetto G, Scorrano L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat Cell Biol. 2011;13:589–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Yang B, Liu Q, Bi Y. Autophagy and apoptosis are regulated by stress on Bcl2 by AMBRA1 in the endoplasmic reticulum and mitochondria. Theor Biol Med Model. 2019;16:18.

    Article  PubMed  PubMed Central  Google Scholar 

  62. Zhou Y, Long Q, Wu H, Li W, Qi J, Wu Y, et al. Topology-dependent, bifurcated mitochondrial quality control under starvation. Autophagy. 2020;16:562–74.

    Article  PubMed  Google Scholar 

  63. Maycotte P, Marín-Hernández A, Goyri-Aguirre M, Anaya-Ruiz M, Reyes-Leyva J, Cortés-Hernández P. Mitochondrial dynamics and cancer. Tumour Biol. 2017;39:1010428317698391.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Medical Center for Human Reproduction, Beijing Chao-Yang Hospital, Capital Medical University, No. 8 Gongtinan Road, Chaoyang District, Beijing, 100020, People’s Republic of China

    Yu-Ting Wan, Shan Liu, Shan-Ke Zhao, Yi-Yang Luo, Ya-Su Lv, Dan-Ni Qu, Ming-Hui Liu & Yuan Li

Authors
  1. Yu-Ting Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shan-Ke Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yi-Yang Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ya-Su Lv
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dan-Ni Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ming-Hui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuan Li.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

(DOCX 16 kb)

ESM 2

(DOCX 19 kb)

ESM 3

(DOCX 30 kb)

ESM 4

(DOCX 30 kb)

ESM 5

(DOCX 19 kb)

ESM 6

(DOCX 19 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wan, YT., Liu, S., Zhao, SK. et al. Effect of luteinizing hormone concentration on transcriptome and subcellular organelle phenotype of ovarian granulosa cells. J Assist Reprod Genet 38, 809–824 (2021). https://doi.org/10.1007/s10815-021-02066-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-021-02066-8

Keywords

Search

Navigation