Compositions and methods for enhancing bioenergetic status in female germ cells

#Since the early 1950s, clinical management of problems associated with ovarian insufficiency and failure, including infertility due to aging or insults, has been restricted by the belief that the pool of oocytes set forth at birth is not amenable to replacement or renewal (Zuckerman, Recent Prog Horm Res 1951 6:63-108). In other words, any therapeutic intervention had to conform to manipulation of the existing stockpile of oocyte-containing follicles to produce a desired clinical outcome. In 2004, however, studies with mice challenged the idea of a fixed ovarian reserve of oocytes being endowed at birth (Johnson et al., Nature 2004 428:145-150). Based on results from several experimental approaches, it was concluded that ovaries of adult female mammals retain rare germline or oogonial stem cells (OSCs) that routinely produce new oocytes in a manner analogous to germline stem cell support of sperm production in the adult testis (Spradling, Nature 2004 428:133-134). Several years later, OSCs were successfully isolated from neonatal and adult mouse ovaries (Zou et al., Nat Cell Biol 2009 11:631-636; Pachiarotti et al., Differentiation 2010 79:159-170). Collectively, these investigations, along with several other reports from studies of mice (Johnson et al., Cell 2005 122:303-315; Wang et al., Cell Cycle 2010 9:339-349; Niikura et al., Aging 2010 2:999-1003) have conceptually validated the use of OSCs as agents for transplantation and as targets for new therapies to modulate ovarian function and female fertility (Tilly et al., Biol Reprod 2009 80:2-12; Tilly et al., Mod Hum Reprod 2009; 15:393-398). In addition, the identification of dormant OSCs in atrophic ovaries of aged mice, which spontaneously resume oocyte formation when exposed to a young adult ovarian environment, indicates that ovarian aging may be reversible (Niikura et al., Aging 2009 1:971-978; Massasa et al., Aging 2010; 2:1-2). The clinical utility of OSCs is now further confirmed by evidence shown herein that a comparable population of oocyte-producing stem cells exists in, and can be purified from, ovaries of healthy reproductive-age women.

#Although these new studies indicate that oocyte numbers in adult ovaries are amenable to therapeutic expansion through OSC-based technology, ovarian aging and failure is determined by both a decline in oocyte number as well as a decline in the quality of the oocytes present in the ovaries. Hence, it is imperative to identify methods for improving oocyte quality, especially in women of advancing maternal age. During the past few decades, because of cultural and social changes, women in the developed world have significantly delayed childbirth. For example, first birth rates for women 35-44 years of age in the United States have increased by more than 8-fold over the past 40 years (Ventura Vital Health Stat 2009 47:1-27; Matthews, NCHS Data Brief 2009 21:1-8). It is well known that pregnancy rates in women at 35 or more years of age are significantly lower, both naturally and with assisted reproduction. The decline in live birth rate reflects a decline in response to ovarian stimulation by gonadotropin hormones (follicle-stimulating hormone or FSH, and luteinising hormone or LH), reduced oocyte and embryo quality and pregnancy rates, and an increased incidence of miscarriages and fetal aneuploidy. In fact, aging-associated chromosomal and meiotic spindle abnormalities in eggs are considered the major factors responsible for the increased incidence of infertility, fetal loss (miscarriage) and conceptions resulting in birth defects—most notably trisomy 21 or Down syndrome—in women at advanced reproductive ages (Henderson et al., Nature 1968 218:22-28; Hassold et al., Hum Genet 1985 70:11-17; Battaglia et al., Hum Reprod 1996 11:2217-2222; Hunt et al., Trends Genet 2008 24:86-93). Although the occurrence and consequences of aging-related aneuploidy in oocytes of humans and animal models have been extensively studied (Tarín et al., Biol Reprod 2001 65:141-150; Pan et al., Dev Biol 2008 316:397-407; Duncan et al., Biol Reprod 2009 81:768-776), approaches to maintain fidelity of chromosome segregation during meiotic cell division with age have remained elusive. At present there is no known intervention to improve the pregnancy outcome of older female patients. In animal studies, chronic administration of pharmacologic doses of anti-oxidants during the juvenile period and throughout adult reproductive life has been reported to improve oocyte quality in aging female mice (Tarín et al., Mol Reprod Dev 2002 61:385-397). However, this approach has significant long-term negative effects on ovarian and uterine function, leading to higher fetal death and resorptions as well as decreased litter frequency and size in treated animals (Tarín et al., Theriogenology 2002 57:1539-1550). Thus, clinical translation of chronic anti-oxidant therapy throughout reproductive life for maintaining or improving oocyte quality in aging females is impractical.

#Mitochondrial dysfunction has a major role in reproductive senescence and, therefore, reproductive function in older women might be improved by the use of mitochondrial nutrients (Bentov et al., Fertil Steril 2010 93:272-275). Aging and age-related pathologies are frequently associated with loss of mitochondrial function, due to decreased mitochondrial numbers (biogenesis and mitophagy), increased aggregation of mitochondria, diminished mitochondrial activity (production of ATP, which is the main source of energy for cells) and mitochondrial membrane potential and/or accumulation of mitochondrial DNA (mtDNA) mutations and deletions. As oocytes age and oocyte mitochondrial energy production decreases, many of the critical processes of oocyte maturation required to produce a competent egg, especially nuclear spindle activity and chromosomal segregation, become impaired (Bartmann et al., J. Assist Reprod Genet 2004 21:79-83; Wilding et al., Zygote 2005 13:317-23). Nicotinamide adenine dinucleotide (NAD+) is a small molecule regulator of many other processes including signaling pathways, cell-cell communication, and epigenetic changes. Once thought to be very stable, levels of NAD+ rise in response to dieting and exercise. Increased NAD+ levels are also associated with the diet known as caloric restriction (CR), which is known to delay numerous aspects of aging and diseases, including infertility (Sinclair Mech Ageing Dev 2005 26:987; Selesniemi et al. Aging Cell 7:622-629, 2008).

#The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying figures, incorporated herein by reference.

#The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of necessary fees.

#FIG. 1 depicts validation of a fluorescence activated cell sorting (FACS)-based protocol for OSC isolation. In FIG. 1a, immunofluorescence analysis of VASA expression (green with blue DAPI counterstain) is shown in adult mouse ovaries using antibodies against the NH2 or COOH terminus of VASA (scale bars, 50 μm). In FIG. 1b, immunomagnetic sorting of dispersed mouse ovaries or isolated oocytes is shown using antibodies against the NH2 or COOH terminus of VASA. Fraction 1 contains cells plus beads prior to separation, Fraction 2 is a wash or flow-through fraction (non-immunoreactive) and Fraction 3 is a bead fraction (VASA-positive cells, highlighted by white arrows). In FIG. 1c, FACS analysis of live or permeabilized cells from dispersed mouse ovaries using antibodies against the NH2 or COOH terminus of VASA is shown. Viable VASA-positive cells are only detected with the COOH antibody (red dashed box) whereas permeabilization enables isolation of VASA-positive cells using the NH2 antibody (blue dashed box). In FIG. 1d, permeabilization of viable VASA-positive cells (red dashed box) obtained with the COOH antibody enables re-isolation of the same cells by FACS using the NH2 antibody (blue dashed box). In FIG. 1e, a schematic representation of the FACS protocols employed using the VASA-COOH antibody for isolation of viable OSCs is shown. FIG. 1f depicts gene expression analysis of germline markers Blimp1 (also referred to as PR domain containing 1 with ZNF domain or Prdm1), Stella, Fragilis (also referred to as interferon induced transmembrane protein 3 or Ifitm3), Tert (telomerase reverse transcriptase), Vasa, Dazl (deleted in azoospermia like) and oocyte markers Nobox (newborn ovary homeobox), Zp3 (zona pellucida glycoprotein 3), Gdf9 (growth differentiation factor 9) in each cell fraction produced during the ovarian dispersion process to obtain cells for FACS-based isolation of OSCs using the VASA-COOH antibody (+ve, VASA-positive viable cell fraction after FACS; −ve, VASA-negative viable cell fraction after FACS; No RT, PCR of RNA sample without reverse transcription; β-actin, sample loading control).

#Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control.

#The term “administration” or “administering” includes routes of introducing a compound(s) to a subject to perform their intended function. Examples of routes of administration that can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, and transdermal. The pharmaceutical preparations are, of course, given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, or inhalation. Oral administration is preferred. The injection can be bolus or can be continuous infusion. Depending on the route of administration, the compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function.

#The agent can be administered alone, or in conjunction with either another agent as described above (e.g. another bioenergetic agent) or with a pharmaceutically-acceptable carrier, or both. The compound can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, the compound can also be administered in a proform which is converted into its active metabolite, or more active metabolite in vivo.