Publications

Zinc Maintains Prophase I Arrest in Mouse Oocytes Through Regulation of the MOS-MAPK Pathway.

Zinc Maintains Prophase I Arrest in Mouse Oocytes Through Regulation of the MOS-MAPK Pathway.

Biol Reprod. 2012;87(1):11

Authors: Kong BY, Bernhardt ML, Kim AM, O'Halloran TV, Woodruff TK

Abstract

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.

PMID: 22539682 [PubMed - in process]

Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro.

Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro.

Hum Reprod. 2012 Jun;27(6):1801-10

Authors: Hornick JE, Duncan FE, Shea LD, Woodruff TK

Abstract

BACKGROUND: In vitro follicle growth is a promising fertility preservation strategy in which ovarian follicles are cultured to produce mature and fertilization-competent oocytes. However, in primates, there has been limited success with in vitro follicle growth starting from primordial and primary follicles because adequate isolation methods and culture strategies have not been established. Understanding how to use primordial follicles for fertility preservation has significant implications because these follicles are the most abundant in the ovary, are found in all females and are fairly resistant to cryopreservation and chemotherapeutics.

METHODS: In the primate ovary, primordial follicles are concentrated near the collagen-rich ovarian cortex. To obtain these follicles, we separated the ovarian cortex prior to enzymatic digestion and enriched the primordial follicle concentration by using a novel double filtration system. To test the hypothesis that a rigid physical environment, as found in vivo, is optimal for survival, primordial follicles were cultured in different concentrations of alginate for up to 6 days. Follicle survival and morphology were monitored throughout the culture.

RESULTS: We found that primate ovarian tissue can be maintained for up to 24 h at 4°C without compromising tissue or follicle health. Hundreds of intact and viable primordial follicles were isolated from each ovary independent of animal age. Follicle survival and morphology were more optimal when follicles were cultured in 2% alginate compared with 0.5% alginate.

CONCLUSIONS: By mimicking the rigid ovarian environment through the use of biomaterials, we have established conditions that support primordial follicle culture. These results lay the foundations for studying the basic biology of primordial follicles in a controlled environment and for using primordial follicles for fertility preservation methods.

PMID: 22456922 [PubMed - in process]

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes.

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes.

Biol Reprod. 2012 Apr;86(4):114

Authors: Bernhardt ML, Kong BY, Kim AM, O'Halloran TV, Woodruff TK

Abstract

Precise coordination of meiotic progression is a critical determinant of an egg's capacity to be fertilized successfully, and zinc has emerged as a key regulatory element in this process. An early manifestation of a regulatory role for this transition metal is the significant increase in total intracellular zinc. This accumulation is essential for meiotic progression beyond telophase I and the establishment of meiotic arrest at metaphase II. The subsequent developmental event, fertilization, induces a rapid expulsion of labile zinc that is a hallmark event in meiotic resumption. In the present study, we show that the zinc fluxes work, in part, by altering the activity of the cytostatic factor (CSF), the cellular activity required for the establishment and maintenance of metaphase II arrest in the mature, unfertilized egg. We propose a model in which zinc exerts concentration-dependent regulation of meiosis through the CSF component EMI2, a zinc-binding protein. Together, the data support the conclusion that zinc itself, through its interaction with EMI2, is a central component of the CSF.

PMID: 22302686 [PubMed - in process]

Inhibin α-subunit N terminus interacts with activin type IB receptor to disrupt activin signaling.

Inhibin α-subunit N terminus interacts with activin type IB receptor to disrupt activin signaling.

J Biol Chem. 2012 Mar 9;287(11):8060-70

Authors: Zhu J, Lin SJ, Zou C, Makanji Y, Jardetzky TS, Woodruff TK

Abstract

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin β-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LβT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LβT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.

PMID: 22267736 [PubMed - indexed for MEDLINE]

Improved fertility preservation care for male patients with cancer after establishment of formalized oncofertility program.

Improved fertility preservation care for male patients with cancer after establishment of formalized oncofertility program.

J Urol. 2012 Mar;187(3):979-86

Authors: Sheth KR, Sharma V, Helfand BT, Cashy J, Smith K, Hedges JC, Köhler TS, Woodruff TK, Brannigan RE

Abstract

PURPOSE: Survival to reproductive age among men with cancer has steadily increased and yet cancer therapy and cancer itself may carry the risk of infertility. Since 2006, we have used a formalized fertility preservation program with expedited fertility care at our institution. We assessed the impact of this program by comparing the frequency of sperm cryopreservation and patient characteristics before and after its implementation.

MATERIALS AND METHODS: Men 18 to 55 years old diagnosed with cancer at our institution from 2002 to 2010 were included in our study. We retrospectively reviewed patient charts to identify those who were offered and subsequently used fertility preservation services before and after program formalization.

RESULTS: From 2002 to 2010 at our institution 4,818 men 18 to 55 years old were diagnosed with cancer, of whom 411 were offered fertility preservation consultation and 249 underwent sperm cryopreservation. Since program implementation, the annual number of men receiving fertility preservation consultation and undergoing sperm cryopreservation increased by 2.4 and 2.7-fold, respectively, while the total number diagnosed with cancer remained fairly constant. Upon substratifying patients into the more conventional reproductive age range of 18 to 40 years 23.4% of all men with cancer in this group were offered consultation before formalization vs 43.3% after formalization (p <0.05). The overall sperm use and discard rates were 8.4% and 14.8%, respectively.

CONCLUSIONS: A formalized institutional fertility preservation program significantly increased the overall number and percent of male patients with cancer who received fertility preservation consultation and pursued sperm cryopreservation. These increases were seen in men with all types of cancer and across all demographics assessed at our institution.

PMID: 22264454 [PubMed - indexed for MEDLINE]

Creating a continuum of care: integrating obstetricians and gynecologists in the care of young cancer patients.

Creating a continuum of care: integrating obstetricians and gynecologists in the care of young cancer patients.

Clin Obstet Gynecol. 2011 Dec;54(4):619-32

Authors: Kong BY, Skory RM, Woodruff TK

Abstract

Cancer therapy can be lifesaving but significantly diminish female reproductive potential. This review provides an overview of the deleterious effects of cancer treatments on reproductive function, the fertility preservation options currently available for young women, and the outcomes of pregnancy after cancer treatment. In addition, special considerations for women who are diagnosed with cancer during pregnancy are discussed. To optimize the continuum of care for the patient, new corridors of communication between obstetricians, gynecologists, and oncology specialists must be developed to ensure the best outcomes for the patient, both in terms of cancer treatment and fertility preservation.

PMID: 22031251 [PubMed - indexed for MEDLINE]

The Gynecologist Has a Unique Role in Providing Oncofertility Care to Young Cancer Patients.

The Gynecologist Has a Unique Role in Providing Oncofertility Care to Young Cancer Patients.

US Obstet Gynecol. 2011 Jan 1;6(1):24-34

Authors: Duncan FE, Jozefik JK, Kim AM, Hirshfeld-Cytron J, Woodruff TK

Abstract

Facing a cancer diagnosis at any age is devastating. However, young cancer patients have the added burden that life-preserving cancer treatments, including surgery, chemotherapy, and radiotherapy, may compromise their future fertility. The possibility of reproductive dysfunction as a consequence of cancer treatment has a negative impact on the quality of life of cancer survivors. The field of oncofertility, which merges the clinical specialties of oncology and reproductive endocrinology, was developed to explore and expand fertility preservation options and to better manage the reproductive status of cancer patients. Fertility preservation for females has proved to be a particular challenge because mature female gametes are rare and difficult to acquire. The purpose of this article is to provide the gynecologist with a comprehensive overview of how cancer treatments affect the female reproductive axis, delineate the diverse fertility preservation options that are currently available or being developed for young women, and describe current measures of ovarian reserve that can be used pre- and post-cancer treatment. As a primary care provider, the gynecologist will likely interact with patients throughout the cancer care continuum. Thus, the gynecologist is in a unique position to join the oncofertility team in providing young cancer patients with up-to-date fertility preservation information and referrals to specialists.

PMID: 21927621 [PubMed - as supplied by publisher]

Nonmalignant diseases and treatments associated with primary ovarian failure: an expanded role for fertility preservation.

Nonmalignant diseases and treatments associated with primary ovarian failure: an expanded role for fertility preservation.

J Womens Health (Larchmt). 2011 Oct;20(10):1467-77

Authors: Hirshfeld-Cytron J, Gracia C, Woodruff TK

Abstract

Cancer treatments can be detrimental to fertility; recent literature has focused on the efforts of fertility preservation for this patient population. It should be recognized, however, that several nonmalignant medical conditions and therapeutic interventions could be similarly hazardous to fertility. Some of these nonmalignant diseases and their treatments that can adversely impact the reproductive axis are gastrointestinal diseases, rheumatologic disorders, nonmalignant hematologic conditions, neurologic disorders, renal disorders, gynecologic conditions, and metabolic diseases. Their negative effects on reproductive function are only now being appreciated and include impaired ovarian function, endocrine function, or sexual function and inability to carry a pregnancy to term. Complications and comorbidities associated with certain diseases may limit the success of established fertility preservation options. Recent advances in fertility preservation techniques may provide these patients with new options for childbearing. Here, we review several fertility-threatening conditions and treatments, describe current established and experimental fertility preservation options, and present three initiatives that may help minimize the adverse reproductive effects of these medical conditions and treatments by raising awareness of the issues and options: (1) increase awareness among practitioners about the reproductive consequences of specific diseases and treatments, (2) facilitate referral of patients to fertility-sparing or restorative programs, and (3) provide patient education about the risk of infertility at the time of diagnosis before initiation of treatment.

PMID: 21827325 [PubMed - indexed for MEDLINE]

Animal age, weight and estrus cycle stage impact the quality of in vitro grown follicles.

Animal age, weight and estrus cycle stage impact the quality of in vitro grown follicles.

Hum Reprod. 2011 Sep;26(9):2473-85

Authors: Hirshfeld-Cytron JE, Duncan FE, Xu M, Jozefik JK, Shea LD, Woodruff TK

Abstract

BACKGROUND: Ovarian tissue cryopreservation is an emerging fertility preservation option, and culturing follicles isolated from this tissue to obtain mature gametes may ultimately be the best solution for patients for whom transplantation is contraindicated. It is unclear, however, how patient-specific variables (including age, weight and menstrual cycle stage) impact follicle growth and quality during three-dimensional culture.

METHODS: We used a mouse model to systematically determine how these variables impact in vitro follicle growth. We characterized metabolic and hormonal profiles of mice at specific ages, weights and cycle stages and secondary follicles from these cohorts were isolated and cultured. We then assessed follicle survival, growth and function, as well as meiotic competence and spindle morphology of the resulting oocytes.

RESULTS: We found that older mice and mice with increased body weight had higher serum cholesterol, abnormal glucose tolerance and lower levels of circulating Anti-Müllerian hormone compared with younger and leaner controls. Secondary follicles isolated from different cohorts and grown in vitro had indistinguishable growth trajectories. However, the follicles isolated from older and heavier mice and those in diestrus had altered hormone profiles. These follicles contained oocytes with reduced meiotic competence and produced oocytes with greater spindle defects.

CONCLUSIONS: These results suggest that the original physical environment of the follicle within the ovary can impact its function when isolated and cultured. These findings are valuable as we begin to use in vitro follicle growth technology for a heterogeneous fertility preservation patient population.

PMID: 21669966 [PubMed - indexed for MEDLINE]

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

Mol Cell Endocrinol. 2011 Aug 6;342(1-2):64-72

Authors: Kim SY, Zhu J, Woodruff TK

Abstract

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production.

PMID: 21664424 [PubMed - indexed for MEDLINE]