A.M. Zamah

Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor, amphiregulin

BACKGROUND The LH surge promotes ovulation via activation of multiple signaling networks in the ovarian follicle. Studies in animal models have shown the importance of LH-induced activation of the epidermal growth factor (EGF)signaling network in critical peri-ovulatory events. We investigated the biological significance of regulatory mechanisms mediated by EGF-like growth factors during LH stimulation in humans. METHODS We characterized the EGF signaling network in mature human ovarian follicles using in vivo and in vitro approaches. Amphiregulin (AREG) levels were measured in 119 follicular fluid (FF) samples from IVF/ICSI patients. Biological activity of human FF was assessed using in vitro oocyte maturation, cumulus expansion and cell mitogenic assays. RESULTS AREG is the most abundant EGF-like growth factor accumulating in the FF of mature follicles of hCG-stimulated patients. No AREG was detected before the LH surge or before hCG stimulation of granulosa cells in vitro, demonstrating that the accumulation of AREG requires gonadotrophin stimulation. Epiregulin and betacellulin mRNA were detected in both human mural and cumulus granulosa cells, although at significantly lower levels than AREG. FF from stimulated follicles causes cumulus expansion and oocyte maturation in a reconstitution assay. Immunodepletion of AREG abolishes the ability of FF to stimulate expansion (P < 0.0001) and oocyte maturation (P < 0.05), confirming the biological activity of AREG. Conversely, mitogenic activity of FF remained after depletion of AREG, indicating that other mitogens accumulate in FF. FF from follicles yielding an immature germinal vesicle oocyte or from an oocyte that develops into an aberrant embryo contains lower AREG levels than that from follicles yielding a healthy oocyte (P = 0.008). CONCLUSIONS EGF-like growth factors play a role in critical peri-ovulatory events in humans, and AREG accumulation is a useful marker of gonadotrophin stimulation and oocyte competence.

Unique Transcriptome, Pathways, and Networks in the Human Endometrial Fibroblast Response to Progesterone in Endometriosis

Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P4) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P4, we compared early (6-h), intermediate (48-h), and late (14-Day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESF) from women with endometriosis (hESFendo) with hESF from women without endometriosis (hESFnonendo). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESF were isolated and treated with P4 (1 μM) plus estradiol (E2) (10 nM), E2 alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P4 in both hESFnonendo and hESFendo, although a blunted response to P4 was observed in the latter. The normal response of hESF to P4 involves a tightly regulated kinetic cascade involving key components in the P4 receptor and MAPK signaling pathways that results in inhibition of E2-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESFendo early response to P4. The abnormal response of this cell type to P4 may contribute to compromised embryonic implantation and infertility in women with endometriosis.

Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle

ABSTRACT Luteinizing hormone (LH) regulation of the epidermal growth factor (EGF) network is critical for oocyte maturation and the ovulatory process. Recent studies have indicated that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR2) play an important role in the control of meiotic arrest. Here, we investigated the involvement of the EGF network in the LH-dependent regulation of the CNP/NPR2 axis and cGMP accumulation. LH/hCG treatment causes a major decrease in both cGMP and the CNP precursor (natriuretic peptide precursor C [Nppc]) mRNA accumulation in vivo and in vitro. However, the cGMP downregulation precedes the decrease in Nppc mRNA by more than 1 h. Amphiregulin, an EGF-like factor, suppresses Nppc mRNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by the EGF receptor (EGFR) kinase inhibitor AG1478. However, the LH-dependent suppression of Nppc is insensitive to AG1478. Similarly, Nppc suppression by LH occurs in follicles from EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP, but is not necessary for LH action. When cGMP concentration in the follicle is measured, the short-term, but not long-term, LH effects on cGMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. Human CG decreases the CNP-dependent cGMP synthesis in wild-type and EGFR knockdown cumulus-oocyte complexes. These findings demonstrate that redundant pathways are involved in the regulation of cGMP. EGFR-dependent events are involved in the short-term regulation of cGMP, whereas the long-term effects may involve regulation of the CNP.

Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

Prepubertal boys treated with high‐dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence‐activated cell sorting using SSEA‐4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA‐4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.

Amphiregulin promotes the maturation of oocytes isolated from the small antral follicles of the rhesus macaque

BACKGROUND In non-primates, the epidermal growth factor (EGF) and EGF-related ligands such as amphiregulin (AREG) serve as critical intermediates between the theca/mural cells and the cumulus-oocyte-complex (COC) following the mid-cycle LH surge. Studies were designed in primates (1) to analyze AREG levels in follicular fluid (follicular fluid) obtained from pre-ovulatory follicles, as well as (2) to assess dose-dependent effects of AREG on oocytes from small antral follicles (SAFs) during culture, including meiotic and cytoplasmic maturation. METHODS Controlled ovulation protocols were performed on rhesus monkeys (n=12) to determine AREG content within the single, naturally selected dominant follicle after an ovulatory stimulus. Using healthy COCs (n=271) obtained from SAFs during spontaneous cycles (n=27), in vitro maturation (IVM) was performed in the absence or presence of physiological concentrations of AREG (10 or 100 ng/ml) with or without gonadotrophins (FSH, 75 mIU/ml; LH, 75 mIU/ml). At the end of the culture period, oocyte meiotic maturation was evaluated and ICSI was performed (n=111), from which fertilization and early embryo development was followed in vitro. RESULTS AREG levels in follicular fluid from pre-ovulatory follicles increased (P<0.05) following an ovulatory bolus of hCG at 12, 24 and 36 h post-treatment. At 12 h post-hCG, AREG levels in follicular fluid ranged from 4.8 to 121.4 ng/ml. Rhesus macaque COCs incubated with 10 ng/ml AREG in the presence of gonadotrophins displayed an increased percentage of oocytes that progressed to the metaphase II (MII) stage of meiosis (82 versus 56%, P<0.05) and a decreased percentage of metaphase I (MI) oocytes (2 versus 23%, P<0.05) relative to controls, respectively. The percentage of either MI or MII oocytes at the end of the culture period was not different between oocytes cultured with 100 ng/ml AREG or in media alone. Fertilization and first cleavage rates obtained by ICSI of all IVM MII oocytes were 93 and 98%, respectively, and did not vary among treatment groups. Of the MII oocytes that fertilized (n=103), 37 were randomly selected and maintained in culture to assess developmental potential. A total of 13 early blastocysts were obtained, with four embryos developing to expanded blastocysts. CONCLUSIONS These data indicate that AREG levels increase in rhesus macaque pre-ovulatory follicles after an ovulatory stimulus, and a specific concentration of AREG (10 ng/ml) enhances rhesus macaque oocyte nuclear maturation but not cytoplasmic maturation from SAFs obtained during the natural menstrual cycle. However, owing to the small number of samples in some treatment groups, further studies are now required.

Will imatinib compromise reproductive capacity

Imatinib mesylate is the first in a family of highly effective, minimally toxic, targeted agents used widely to treat Philadelphia-positive leukemias and selected other cancers, leading to a steady rise in the prevalence of patients using such therapy. Because failure of therapy would require conventional gonadotoxic chemotherapeutics, many female patients using imatinib may choose to preserve fertility. Herein, we provide evidence of a potential negative effect of imatinib on ovarian function by reporting the first case of a woman who showed a severely compromised ovarian response to gonadotropin stimulation while on imatinib, with a normal ovarian response after stopping this medication.

Correction to Multiple Pathways Mediate Luteinizing Hormone Regulation of cGMP Signaling in the Mouse Ovarian Follicle. [Biol Reprod 91, 1, (2014). 1-11] DOI: 10.1095/biolreprod.113.116814

Xueqing Liu, Fang Xie, Alberuni Musa Zamah, Binyun Cao, and Marco Conti College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China Department of Obstetrics, Gynecology and Reproductive Sciences, Center for Reproductive Sciences, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco (UCSF) Medical Center, UCSF, San Francisco, California