Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium

Abstract

Purpose: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mediate intracellular functions by binding their specific protein G-coupled gonadotrophin receptor, respectively FSH receptor (FSHR) and LH/choriogonadotrophin receptor (LHCGR). Whereas the expression of FSHR and LHCGR in mammals was considered gonad-specific and cell-specific, studies identified gonadotrophin receptors in human female extragonadal reproductive tissues. This study aims to demonstrate that gonadotrophin receptors are expressed in endometrium and mediates intracellular functions.

Methods: Collected endometria (n = 12) from healthy patients (mean age of 36 ± 6) were primary cultured for 24 h. The presence of gonadotrophin receptors was evaluated by RT-PCR followed by the sequencing of the resulted amplicons and by immunohistochemistry in original samples. Endometrial primary cultures were treated with increasing concentration (range 0-100 ng/ml) of either recombinant human LH (rhLH) or recombinant human FSH (rhFSH). Endometria controls had gonadotrophin replaced by the same volume of the culture medium. In gonadotrophin-treated samples, it was evaluated the intracellular cyclic adenosine monophosphate (cAMP) content by enzymatic immunoassay and the expression of steroidogenic genes by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).

Results: The sequencing of the RT-PCR amplicons confirmed the presence of both gonadotrophin receptors and immunohistochemistry localized them on the membrane of endometrial glands cells throughout the glandular epithelium. The gonadotrophin-receptor complex was able to increase the intracellular cAMP in a dose-response and time-course manner and to induce steroidogenic genes expression.

Conclusion: This study demonstrates that both gonadotrophin receptors are expressed along the glandular epithelium of endometria and they mediate the effects of gonadotrophins on intracellular functions.

Keywords: Endometrium; FSHR; Gonadotrophin; LHCGR; Steroidogenic genes.

Fig. 1

Lines 1–3 refer to samples of original endometria recovered from three different patients. The template was omitted from the negative control (−). M DNA standard ladder, hGCs human granulosa cells, IPLB insect cell line

Fig. 2

Nucleotide sequences of a LHCGR and b FSHR cDNA. Lines are namely according to the resulted sequence names’ or with the accession number of corresponding sequence. Asterisks underneath alignments mark the matching between sequences

Fig. 3

a LHCGR are depicted in red color b higher magnification. c FSHR are depicted in green color; d higher magnification. Controls in which the primary Abs were omitted were negative (not shown)

Fig. 4

Representative (n = 1) dose-response production of intracellular cAMP in primary cultured endometria collected during the proliferative phase of menstrual cycle stimulated by increasing concentrations of rhLH or rhFSH, measured after 2 h of incubation in the presence of 5 μM IBMX. The cAMP measurement were conducted in duplicate and expressed as mean. Bars represent the two values used for to calculate the mean value. Significant differences between treated cases and untreated controls were marked by * (P < 0.001), Kruskal-Wallis test followed by the Dunn-Bonferroni’s test

Fig. 5

Intracellular cAMP production in primary cultures of endometrium induced by 10 ng/ml of rhLH or rhFSH in time-course experiment (n = 4). The cAMP measurements were conducted in duplicate and expressed as mean and standard deviation. Significant differences between treated cases and untreated controls were marked by * (P < 0.001), Kruskal-Wallis test followed by the Dunn-Bonferroni’s test

Fig. 6

Gonadotrophin-stimulated intracellular cAMP content expressed as fold change in comparison to untreated control proliferative endometria set arbitrarily to 1 (n = 6). Significant differences versus untreated controls were marked by * (P < 0.001), Kruskal-Wallis test followed by the Dunn-Bonferroni’s test

Fig. 7

Aromatase CYP19A1 and cytochrome P450scc expression in endometria collected during the proliferative phase of the menstrual cycle. Lines 1–3 refer to individual original samples obtained from three different patients. M DNA standard ladder

Fig. 8

a Aromatase CYP19A1 and b cytochrome P450scc gene expressions in primary culture of proliferative endometria after 24-h incubation with 10 ng/ml of gonadotrophins (n = 6). Negative controls were cultured only with culture medium. Significant differences between treated samples and untreated controls were marked by * (P < 0.001), Kruskal-Wallis test followed by the Dunn-Bonferroni’s test