Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice

Abstract

Objective: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts.

Design: Xenotransplantation model.

Setting: University-based research laboratory.

Patients/animals: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice.

Interventions: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 μg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries.

Main outcome measures: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature.

Results: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 μg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 μg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization.

Conclusions: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

Keywords: Insulin-like growth factor 1; endothelial cell cotransplantation; folliculogenesis; growth hormone; human cortical xenograft.

FIGURE 1

Sustained paracrine stimulus of xenograft-resident follicles with exogenous insulin-like growth factor 1 (IGF1). (A to C) Cryosections from xenografts of human ovarian cortical tissue were labeled with an antibody specific for IGF1 receptor; primary (A), secondary (A), and antral (B and C) stage follicles were positive for IGF1 receptor on the surface of granulosa (A to C) and theca (B and C) cells. The antral follicle in (c) was colabeled with the fluorescently conjugated lectin (Griffonia simplicifolia) and antibodies against phosphorylated Akt and follicle-stimulating hormone receptor. (D) Schematic representation of the experimental framework for cotransplantation of ovarian cortical strip along with either control or IGF1-expressing endothelial cells (ECs) in immune-compromised mice. (E) Cell culture supernatant from ECs transduced with lentiviral particles encoding IGF1 cDNA shows an approximately 100-fold increased presence of IGF1 in the supernatant of transduced vs. control ECs. (F) ECs, labeled by mCherry fluorescent protein persist in the margins of long-term xenografts. Error bars in (E) represent standard deviation between three replicate samples. Scale bars: 100 μm in A to B and F and 1 mm in C.

FIGURE 2

Paracrine insulin-like growth factor 1 (IGF1) stimulus in xenografts promotes follicular advance. (A to F) Xenografts were recovered at 3 (A and B), 8 (C and D), and 14 (E and F) weeks after cotransplantation with control or IGF1-expressing ECs. (G) Intrapatient subanalysis of follicle distribution, where sufficient replicates are available. The percentage of primordial, primary, and secondary follicles was quantified for each xenograft (shown in A, C, and E), with secondary follicles separated into simple and preantral subtypes (shown in B, D, and F). The bars in (A to F) represent the median percentages of follicle subtypes with 95% confidence interval and with each dot representing one replicate; numerical P values are shown. Error bars in (G) represent standard deviation, with asterisks denoting a P value of <.05 (*) or <.01 (**).

FIGURE 3

Paracrine insulin-like growth factor 1 (IGF1) stimulus affects the distribution of antral follicles and corpora lutea in long-term xenografts. (A to C) Histologic examples (A and B) and absolute numbers (C) of antral follicles identified in xenografts cotransplanted with control or IGF1 endothelial cells after 8 (A) or 14 (B and C) weeks. (D to F) Histologic (D) and molecular (E) evidence of corpora lutea-like structures that increased (f) in xenografts cotransplanted with IGF1-expressing endothelial cells. The insets in (D) and (E) are magnified in the panels below. Scale bars: 100 μm.

FIGURE 4

Direct injection of recombinant insulin-like growth factor 1 (IGF1) into the mouse ovary promotes follicle advance and increased oocyte yield. (A to C) Oocytes were recovered from mice and quantified (C) following bilateral ovarian injection of PBS (A) or IGF1 (B)and subsequent hyperstimulation (D to F). Mice were injected intraperitoneally with ethynyl-deoxyuridine at the time of ovary injection with IGF1/PBS (d), with the ovaries harvested for immunofluorescent quantification of proliferating cells 2 days later (E); the percentage of follicle-resident nuclei that were positive for ethynyl-deoxyuridine at across follicles stages is shown in (F) with the number of follicles represented below each bar. Error bars in (C) and (F) represent standard deviation between replicate samples; P value shown in (C) and asterisks in (F) denote a P value of <.05 (*) or <.01 (**). Scale bar: 1 mm.

FIGURE 5

Ribonucleic acid sequencing reveals acute elevation of transcripts evident at later follicle stages. (A to B) The ovaries were recovered from mice for ribonucleic acid sequencing at 24 and 72 hours following direct injection with phosphate-buffered saline (PBS) or recombinant IGF1 (A); a multidimensional scale plot of the relative similarity between samples is shown in (B). (C to D) Heatmaps showing the 53 (C) and 52 (D) transcripts that were differentially expressed (P<.05) between the PBS and IGF1 conditions at 24 (C ) and 72 (D) hours after injection. (E to F) Volcano plots showing the distribution of differentially expressed transcripts between PBS and IGF1 conditions at 24 (E ) and 72 (F) hours after injection; transcripts with relevance to sex steroid biosynthesis/metabolism and/or follicle maturation/luteinization are highlighted in red in (E to F).