WO2024155955A1

WO2024155955A1 - Indole, derivatives, and uses in reproductive medicine

Info

Publication number: WO2024155955A1
Application number: PCT/US2024/012279
Authority: WO
Inventors: Daniel Kalman; Alyson Swimm; Robert SONOWAL; Sam MESIANO
Original assignee: Emory University; Case Western Reserve University
Current assignee: Emory University; Case Western Reserve University
Priority date: 2023-01-20
Filing date: 2024-01-19
Publication date: 2024-07-25
Anticipated expiration: 2025-07-20

Abstract

Disclosed herein are methods of isolating an oocyte or ovum for use in in vitro fertilization comprising contacting a sample comprising an oocyte or ovum with indole or a derivative providing an indole preserved oocyte or ovum. In certain embodiments, this disclosure relates to compositions for preserving or culturing oocytes or ova for further use in reproductive medicine.

Description

INDOLE, DERIVATIVES, AND USES IN REPRODUCTIVE MEDICINE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/440,159 filed January 20, 2023. The entirety of this application is hereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AG054903 and DK074731 awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND

Ova are human eggs distinct from oocytes. Oocytes are ovarian follicle cells that contain an intact nuclear envelope (germinal vesicle) and a plasma membrane surrounded by a second exterior membrane called the zona pellucida. Ovulation of oocytes is triggered by hormones. In a typical in vitro fertilization (IVF) procedure, an ovulation cycle is boosted to produce multiple oocytes by administering gonadotropins, e.g., first a follicle-stimulating hormone (FSH) followed by a luteinizing hormone (LH). Ultrasound-guided methods are used to visually harvest mature follicles wherein oocytes are aspirated, usually using a syringe. Thereafter, concentrated sperm and oocyte are mixed to form an ovum.

As animals age, germ cells exhibit increased aneuploidy and genomic instability, thereby reducing embryo viability and allowing the accumulation of mutations causing birth defects that ultimately limit fecundity and reproductive span. The germline DNA damage response (DDR) comprises cell cycle checkpoint regulators, DNA repair enzymes, and apoptotic proteins that ensure genome integrity. Upon sensing DNA damage, cell cycle checkpoints delay cell division and facilitate decisions to either repair the damage or initiate cell death to eliminate damaged embryos, a process ensuring faithful transmission of genetic information across generations. DDR pathways often become dysregulated with age, raising the possibility that reduced capacity for DNA damage surveillance, repair, or cell death may contribute to increased rates of aneuploidy and reduced fecundity evident in older individuals. Thus, there is a need to identify improved reproductive methods for older individuals.

Sonowal et al. report indoles from commensal bacteria extend healthspan. PNAS, 114, E7506-E7515 (2017).

Mori et al. report cruciferous vegetable intake reduced mortality in middle-aged adults. Clin Nutr, 38, 631-643 (2019).

Ruiz et al. report functional microbiome deficits associated with ageing. Aging Cell, 19, el3063 (2020).

Meng et al., indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells. J Nutr, 130, 2927-2931 (2000).

References reported herein are not an admission of prior art.

SUMMARY

In certain embodiments, this disclosure relates to methods of preserving an oocyte or ovum for use in in vitro fertilization comprising contacting an isolated oocyte or ovum with indole or a derivative, and optionally culturing the oocyte or ovum, providing an indole preserved oocyte or ovum. In certain embodiments, methods further comprise contacting the indole preserved oocyte with sperm providing an embryo and further implanting the embryo in a uterus of a female subject.

In certain embodiments, this disclosure relates to methods of purifying an oocyte for use in inducing the formation of ovum comprising contacting a sample comprising oocytes with indole or derivative providing an indole preserved oocyte, detecting an oocyte that is abnormal, separating the abnormal oocyte providing a purified normal oocyte, and inducing formation of an ovum from the purified normal oocyte.

In certain embodiments, this disclosure relates to methods of improving the fertility of a female subject, oocyte quality, de novo production, or improving the recovery of ovulated oocytes or ova, comprising administering to a female subject an effective amount of an indole or derivative thereby improving fertility in a female subject. In certain embodiments, the indole or derivative is orally or systemically administered to the female subject. In certain embodiments, the indole or derivative is administered locally to the ovary of the female subject. In certain embodiments, methods further comprise obtaining an oocyte or ovum from a female subject and forming a fertilized ovum by in vitro fertilization. In certain embodiments, methods further comprising implanting the fertilized ovum in the female subject. In certain embodiments, the pregnancy outcome of the female subject is improved compared to a control standard.

In certain embodiments, this disclosure relates to compositions comprising an oocyte, or progeny cell and indole or derivative and an agent selected from leuprolide acetate, gonadotropin releasing horm one-agonists, follitropin beta, urofollitropin, follicle stimulating hormone (FSH)), combination of FSH and LH, luteinizing hormone, human chorionic gonadotropin (hCG), or combinations thereof. In certain embodiments, the progeny cell is an ovum.

In certain embodiments, this disclosure relates to compositions comprising an oocyte or progeny cell and indole or derivative. In certain embodiments, the progeny cell is an ovum. In certain embodiments, the composition further comprises nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinic acid, tryptophan, quinolinic acid, fisetin, quercetin, resveratrol, hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin, berberine, or combinations thereof.

In certain embodiments, the composition is a cell culture medium, an oocyte collection solution, an oocyte washing solution, an oocyte in vitro maturation medium, an oocyte in vitro fertilization medium, an embryo medium, vitrification solution, cry opreservation solution, or embryo thawing medium.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figures 1A-1G show data indicating indoles regulate DNA repair and survival of mouse oocytes.

Figure 1A shows a schematic of mouse oocyte quality assessment.

Figure IB shows data on the percentage of tail DNA in the comets of X-irradiated CD1 oocytes from mice treated with indole-3-aldehyde (ICA) or vehicle (n > 70 comets per condition).

Figure 1C shows oocyte number per mouse from young (3 months old) or old (> 6 months) CD1 mice treated with ICA or vehicle.

Figure ID shows data on the frequency of fragmented oocytes. Figure IE shows data on IVF success rate. Values above the bars represent the number of fertilized oocytes/total number of oocytes.

Figure IF shows data on the frequency of developmental arrest observed in embryos after IVF. Values above the bars represent the number of arrested embryos/total embryos scored.

Figure 1G shows data on the frequency of abnormal oocytes from young (3 months old) C57BL/6 mice ICA/vehicle treated for 2 days and subjected to post-ovulatory aging. Values above the bars represent number of abnormal oocytes/total number of oocytes.

Figure 2A illustrates a model of indole quality control regulation. Although it in not intended that embodiments of this disclosure be limited by any particular mechanism, it is believed that indole and the indole derivative (ICA) are derived from the microbiota or from dietary sources (e.g., cruciferous vegetables) and act via AhR, MRN-1, and p53/CEP-l to detect DNA damage and activate the DSB repair machinery when damage is reparable, and cell death via the apoptosis activating factor CED4 when it is not, ensuring genome integrity and homeostasis. Alternatively, dysbiosis, dietary changes, or aging results in decreased levels of indole and thereby increased aneuploidy and genome variability.

Figure 2B illustrates that indoles act in a “quality control” capacity via MRN and p53 to affect genome integrity. In youth or low stressor levels, repair capacity is high, and indole has little effect. With age or increasing stressor levels, indole augments repair together with cell death, so that surviving cells have higher genomic integrity, but more limited diversity compared to cells without indole. Thus, cell death removes heavily damaged cells, limiting their impact on diversity and fecundity. Increasing either repair or cell death increases the percentage of Fl animals with intact genomes, providing a survival advantage in successive generations. Limiting indole, which occurs with dysbiosis, dietary changes, or aging, relaxes the p53 checkpoint, resulting in decreased repair and cell death, increased aneuploidy, and, in worms, more males. Males outcross via sexual reproduction, increasing genomic diversity and allowing rapid adaptation to changing environments. Worm and mammalian oocytes have different set points for death and repair, with worms favoring survival and repair over death and mammals favoring the opposite.

DETAILED DISCUSSION

An “embodiment” is an example of this disclosure and not necessarily limited to such example. It is also to be understood that the terminology used herein is for describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the examples and does not pose a limitation on the scope unless otherwise claimed.

Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The dates of publications provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. Use of the term "about" is intended to describe values either above or below the stated value in a range of approximately plus or minus 10%; in other embodiments the values may range in value either above or below the stated value in a range of approximately plus or minus 5%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

"Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characterise c(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.

As used herein, "subject" refers to any animal, preferably a human patient, livestock, or domestic pet. In certain embodiments, the subject is a female human subject, e.g., 18 years old or older. In certain embodiments, the subject is a female human subject 30 or 40 years old or older.

As used herein, the term "derivative" refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, e.g., replacing an amino group, hydroxyl, or thiol group with a hydrogen, substituted, a salt, in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur atom, or replacing an amino group with a hydroxyl group. The derivative may be a prodrug, alkyl ester, alkanoyl ester, comprise a lipid, polyethylene glycol, saccharide, or polysaccharide.

The terms “indole or derivative,” are contemplated to include indole, indole-3 - carbaldehyde, indole-3 -carbinol, indole propionic acid, or a substituted indole, or a compound of the following formula:

or salt thereof wherein R is hydroxy, alkyl, hydroxy alkyl, formyl, alkanoyl, alkoxy, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, wherein R is optionally substituted.

The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, - NRaSO₂Rb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, - S(=O)₂Ra, -OS(=O)₂Ra and -S(=O)₂ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. The substituents may further optionally be substituted.

The terms, “cell culture” or “growth medium” or “media” refers to a composition that contains components that facilitate cell maintenance and growth through protein biosynthesis, such as vitamins, amino acids, inorganic salts, a buffer, and a fuel, e.g., acetate, succinate, a saccharide and/or optionally nucleotides. Typical components in a growth medium include amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and others); vitamins such as retinol, carotene, thiamine, riboflavin, niacin, biotin, folate, and ascorbic acid; carbohydrate such as glucose, galactose, fructose, or maltose; inorganic salts such as sodium, calcium, iron, potassium, magnesium, zinc; serum; and buffering agents. Additionally, a growth media may contain phenol red as a pH indication. Components in the growth medium may be derived from blood serum or the growth medium may be serum-free. The growth medium may optionally be supplemented with albumin, lipids, insulin and/or zinc, transferrin or iron, selenium, ascorbic acid, and an antioxidant such as glutathione, 2-mercaptoethanol or 1 -thioglycerol. Other contemplated components contemplated in a growth medium include ammonium metavanadate, cupric sulfate, manganous chloride, ethanolamine, and sodium pyruvate. Ova are human eggs distinct from oocytes. Oocytes are cells that contain an intact nuclear envelope (germinal vesicle) and a plasma membrane surrounded by a second exterior membrane called the zona pellucida, all three of which can be distinctly observed using light microscopy. Oocytes divide by meiosis I and arrest at metaphase of meiosis II. Ovum is a haploid female gamete resulting from completion of meiosis II at fertilization.

An “oogonial stem cell (OSC)” refers to a progenitor stem cell of an oocyte. MacDonald report subpopulations of OSCs that are separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Stem Cells Dev, 2023, 32(5-6): 99-114.

When preparing for in vitro fertilization (IVF) oocytes are typically obtained from superovulating subjects. “Superovulation” techniques, such as treatment of a female subject with hormones, used in IVF are designed to stimulate the ovaries to produce several oocytes. The medications required to boost oocyte production may include leuprolide acetate, gonadotropin releasing horm one-agonists, follitropin beta, urofollitropin, follicle stimulating hormone (FSH)), combination of FSH and LH, luteinizing hormone, and/or, human chorionic gonadotropin (hCG).

Collection of oocytes can be performed under transvaginal ultrasound guidance. Often a transfer device such as a needle is inserted (e.g., under IV sedation) through the vaginal wall into the ovaries using ultrasound to locate each follicle. A sample of follicular fluid is isolated and transferred to a container providing isolated oocytes.

Disclosed herein are methods of isolating an oocyte or ovum for use in in vitro fertilization comprising contacting a sample comprising oocyte with indole or derivative providing an indole preserved oocyte or ovum. In certain embodiments, this disclosure relates to composition for preserving or culturing oocytes or ovum for further use in reproductive medicine.

In certain embodiments, this disclosure relates to methods of preserving an oocyte or ovum for use in in vitro fertilization comprising contacting an isolated oocyte or ovum with indole or derivative and storing or culturing the oocyte or ovum providing an indole preserved oocyte or ovum.

In certain embodiments, this disclosure relates to methods of preserving an oocyte or ovum for use in in vitro fertilization comprising contacting an isolated oocyte or ovum with indole or derivative, and optionally culturing the oocyte or ovum, providing an indole preserved ovum. In certain embodiments, methods further comprise contacting the indole preserved oocyte with sperm providing an embryo and further implanting the embryo in a uterus of a female subject. In certain embodiments, the female subject is 18 years of age or older. In certain embodiments, the female subject is 30 or 35 years old or older.

In certain embodiments, this disclosure relates to methods of purifying an oocyte for use in inducing the formation of ovum comprising contacting a sample comprising oocytes with indole or derivative providing an indole preserved oocytes, detecting an oocyte that is abnormal, e.g., due to damaged DNA, separating the abnormal oocyte providing a purified normal oocyte, and inducing formation of an ovum from the purified normal oocyte. In certain embodiments, the female subject is 18 years of age or older In certain embodiments, the female subject is 30 or 35 years old or older.

In certain embodiments, this disclosure relates to methods of improving the fertility of a female subject, oocyte quality, de novo production, improve the recovery of ovulated oocytes comprising administering to a female subj ect an effective amount of an indole or derivative thereby improving fertility in a female subject. In certain embodiments, the indole or derivative is orally or systemically administered to the female subject. In certain embodiments, the indole or derivative is administered locally to the ovary of the female subject. In certain embodiments, methods further comprise obtaining an oocyte or ovum from a female subject and forming a fertilized egg by in vitro fertilization. In certain embodiments, the pregnancy outcome of the female subject is improved compared to a control standard. In certain embodiments, the female subject is 18 years of age or older In certain embodiments, the female subject is 30 or 35 years old or older.

In certain embodiments, this disclosure relates to methods producing an oocyte or ovum for in vitro fertilization comprising transferring a composition comprising indole or derivative and exogenous, autologous, mitochondria into an oocyte of a subject; wherein the exogenous, autologous mitochondria are isolated from an autologous oogonial stem cell (OSC) of the subject or the progeny of an autologous OSC of the subject; and wherein the OSC is a non-embryonic stem cell that is mitotically competent and expresses a stage-specific embryonic antigen; and isolating the oocyte or ovum for use in in vitro fertilization. In certain embodiments, methods further comprise contacting the oocyte or ovum with indole or derivative providing an indole preserved oocyte or ovum. In certain embodiments, the female subject is 18 years of age or older In certain embodiments, the female subject is 30 or 35 years old or older. In certain embodiments, this disclosure relates to methods of preserving oocytes or methods of preparing an oocyte or ovum for in vitro fertilization (IVF) comprising contacting a sample comprising oocyte or ovum with indole or a derivative providing a composition with an indole preserved oocyte or ovum.

In certain embodiments, this disclosure relates to compositions comprising an oocyte, oogonial stem cell (OSC), or progeny cell and indole or derivative and an agent selected from leuprolide acetate, gonadotropin releasing horm one-agonists, follitropin beta, urofollitropin, follicle stimulating hormone (FSH)), combination of FSH and LH, luteinizing hormone, and human chorionic gonadotropin (hCG), or combinations thereof.

In certain embodiments, this disclosure relates to compositions comprising an oocyte, oogonial stem cell (OSC), or progeny cell, and indole or derivative. In certain embodiments, the progeny cell is an ovum. In certain embodiments, the composition further comprises nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinic acid, tryptophan, quinolinic acid, fisetin, quercetin, resveratrol, hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin, berberine, or combinations thereof.

In certain embodiments, the composition is a cell culture medium, an oocyte collection solution, an oocyte washing solution, an oocyte in vitro maturation medium, an oocyte in vitro fertilization medium, an embryo medium, vitrification solution, cryopreservation solution, or embryo thawing medium.

In certain embodiments, cryopreservation methods are contemplated to include cooling the indole preserved oocyte or ovum, optionally in the presence of indole or an indole derivative, to a temperature of less than 32 degrees Fahrenheit providing a frozen indole preserved oocyte or ovum composition. In certain embodiments, cooling the indole preserved oocyte or ovum is to a temperature of less than zero degrees Fahrenheit, or to a temperature of less than negative 20 degrees Fahrenheit, or less than negative 50 degrees Fahrenheit.

In certain embodiments, methods further comprise storing the frozen indole preserved oocyte or ovum for a predetermined amount of time and thereafter warming the frozen indole preserved oocyte or ovum composition providing a thawed oocyte or ovum, optionally culturing the thawed oocyte or ovum, contacting the thawed and/or cultured oocyte or ovum with sperm providing an embryo, and implanting the embryo in the uterus of a female subject. A microbiota and dietary metabolite integrates DNA repair and cell death to regulate embryo viability and aneuploidy during aging

The nematode Caenorhabditis elegans (C. Elegans) eat bacteria, and male frequencies together with embryo lethality serve as readily quantifiable readouts of the presence of an abnormal number of chromosomes in a cell (aneuploidy) on the X chromosome and autosomes, respectively. Animals cannot synthesize indoles and instead rely on dietary components. In mammals, indoles enhance the integrity of the epithelial barrier, thereby limiting dissemination of bacteria and bacterial antigens, reduce deleterious inflammation, and enhance motility in the aged. In C. elegans, indoles also extend the reproductive period, allowing aged animals to produce viable embryos for longer. These molecules may affect germ cell quality. Experiments reported herein indicate that in aging germ cells from both vertebrates and invertebrates, indoles repair DNA damage or initiate cell death, depending on the level of damage, thereby increasing genome integrity in the embryo. In so doing, indoles increase the proportion of viable embryos, which promotes fecundity across generations.

Data presented here indicate that indole regulates cellular repair and cell death mechanisms in worms and mammals to limit aneuploidy and increase genome integrity in the germline, so as to promote high-fidelity transmission of genetic information across generations. These data highlight the importance of an ancient interkingdom signaling molecule in promoting genetic homeostasis or, in its absence, driving genetic variation.

Data presented here indicate that the indoles regulate the sensitivity of the DDR in both meiosis and mitosis to promote genome integrity, limit aneuploidy, and thereby ensure genome quality of daughter cells. In C. elegans, the DDR factors MRE-11, ATM-1, and p53/CEP-l mediate the protective response of indole following radiation, mutation, or aging, in line with previous reports showing that homology-mediated DNA repair via MRN is the primary detection and repair mechanism in the germline. Data on hus-1 mutant suggest an additional role for the 9-1-1 pathway in mediating indole effects. The 9-1-1 pathway and NHEJ mediate DNA repair responses in somatic cells and tissues, particularly in adults and developing embryos, raising the possibility that the effects of indoles act on cells outside the germline.

Protective effects of indole against radiation or aging involve AhR, a phenotype conserved across diverse phyla. In immortalized cells, ionizing radiation acts via H2AX, DNA-protein kinase (PK), and ATM to promote DNA repair, an effect that also depends on AhR. These data are in line with data indicating that TCA acts via AhR to limit apoptosis and promote radioprotection. Indole derivatives promote radioprotection both in vitro and in vivo. Data suggests that indole acts via cellular mechanisms involving AhR interfacing with chromosomal DDR and repair machinery. However, a more precise mechanism by which indole detects DNA damage and induces repair awaits biochemical characterization of indole effects on these processes.

The observation that indole had opposite effects on embryo lethality at 60 Gy versus 120 Gy raised the possibility that indole senses both the level of DNA damage and the cellular repair capacity and thus acts as a rheostat to either initiate repair or induce cell death. Both mechanisms depend on p53, but the cell death pathway additionally depends on CED-4, which encodes the worm homolog of APAF-1, an activator of apoptosis, but does not depend on the CED-3 caspase.

An extensive array of factors and mechanisms exist to stimulate cell death (e.g., caspases, APAF, etc.) or limit it (e.g., cellular inhibitor of apoptosis protein (cIAP), B cell lymphoma-2 (BCL-2), etc.) and to integrate extrinsic or intrinsic signals. Pathways favoring survival over death have been exploited by viruses to enable their unfettered replication without destruction of the host cell, which has likely facilitated the evolution of complex positive and negative signaling factors controlling cell survival. Data presented here suggest that molecules from microbiota and plants may likewise exert regulatory control over the DNA repair pathways to induce cell death on the one hand or repair and survival on the other, depending on the level of DNA damage and the cellular repair capacity. This regulation may be particularly important in allowing adaptation to change in the DNA repair capacity, which may decline with age or, alternatively, damage, which may increase with age. The indole-mediated increase or decrease in embryo lethality improved viability in subsequent generations, highlighting the utility of the indole rheostat in promoting overall fecundity. It is contemplated that indole, together with the DNA damage sensing and response pathways, and apoptotic pathways act as a quality control regulator to ensure genome integrity.

Indoles limit intergenerational embryo lethality and male frequency induced by environmental stressors in C. elegans

The effects of indole on male frequencies and embryo lethality in the progeny of animals subjected to heat stress or X-irradiation were evaluated. Wild-type (N2) worms were exposed to indole or carrier (MeOH) from the embryo stage onward. Animals at the fourth larval (L4) stage were either left unstressed or subjected to transient heat stress and then allowed to deposit embryos (Fl) at the permissive temperature. The sex and viability of their progeny were then enumerated. The indole or carrier treatment was continued for the duration of the experiment in both FO and F 1 generations. Heat stress alone increased the numbers of both males and dead embryos. Under both normal growth conditions and heat stress, indole reduced the number of males and the number of dead embryos. The reduction in male frequencies was too small in magnitude to permit resolution of changes in numbers of diakinetic chromosomes in the proximal gonad, an assay commonly used to assess aneuploidy in him strains. Similar to heat stress, X-irradiation [60 gray (Gy)] increased both the number of male progeny and the number of dead embryos. As with heat stress, exposure to indole reduced the number of dead embryos and the frequency of males. Together, these data indicate that indoles reduce intergenerational aneuploidy associated with environmental stressors.

Indoles limit intergenerational embryo lethality and male frequency due to mutations in C. elegans

Induction of males with stressors reads out X-chromosomal aneuploidy, but embryo lethality may result from other factors besides autosomal nondisjunction. To address this, the effects of indole on male frequency and embryo lethality in "high incidence of males" (him) mutant strains, which produce males and dead embryos due to X chromosome and autosome nondisjunction, was evaluated. The him mutants affect critical stages of chromosomal segregation and DNA repair during meiosis, including chromosomal pairing and attachment to the nuclear envelope (him-8, zim-1, and zim-2); homologous chromosome alignment and synapsis formation (him-3, htp-3, syp-1, and syp-2); homology recognition and double-stranded break (DSB) formation (spo-11, him-19, and him-5); crossover formation, recombination, and resolution of Holliday junction (him-5 and him-6); and DSB repair (him-5). Mutant strains were grown either in indole or in control but without exposure to stressors, and their progeny wwew enumerated. Indole reduced male frequencies in animals with mutations in him-5, him-6, him-19, zim-1, and zim-2, indicating suppression of nondisjunction on the X chromosome. Indole also decreased the number of dead embryos in animals with mutations in htp-3, him-8, and him-5, indicative of an effect of indole in limiting autosomal nondisjunction. Indole was without effect on male frequencies in him-8 mutants, likely because indole did not affect X chromosome pairing in a mutant defective in that process. For similar reasons, indole was likely without effect on embryo lethality in zim-1 or zim-2 mutants, which govern autosomal pairing. In some mutants (e g., syp- 1, syp-2, spo-11, and him-3), indole did not alter male frequencies or embryo viability. However, the number of viable animals was so low as to preclude evaluation of enough animals to achieve significance. As a control, indole did not affect total brood size in N2 or in all mutants except htp- 3(tm3655), syp-l(me!7), and syp-2(ok307). Together, data showing suppression of males or embryo lethality in several him mutants indicate that indole limits nondisjunction on both the X chromosome and autosomes and acts at multiple stages of meiosis.

Indoles act via the DDR pathway to limit aneuploidy and embryo lethality in C. elegans

Both irradiation and indole-suppressible him mutants are associated with DNA strand breaks or repair during recombination, raising the possibility that indole regulates male numbers and embryo viability via DNA damage sensing pathways or the p53/C. elegans p53-like protein-1 (CEP-1) effector. To test this, strains containing inactivating mutations in components of the 9-1- 1, nonhomologous end joining (NHEJ), MRE-11-RAD-50-NBS-1 (MRN) sensors, or CEP-1 effector were grown in the presence or absence of indole and subjected to X-irradiation. The numbers of males and dead embryos in their progeny were enumerated. Indole or carrier treatment was continued for the duration of the experiment in the FO and Fl generations. Indole suppressed male numbers in strains with mutations in hus-1 and cku-70, which encode components of the 9- 1-1 and NHEJ pathways, respectively. However, no suppression was evident in strains containing mutations in mre-11 or atm-1, which encode members of the MRN/Ataxia telangiectasia mutated- 1 (ATM-1) pathway. Indole also reduced embryo lethality in NHEJ pathway mutants, but not in the 9-1-1 and MRN pathway mutants. Together, these data indicate that in response to radiation damage, indole suppresses nondisjunction on the X chromosome via the MRN pathway and limits embryo lethality via both the MRN and 9-1-1 pathways.

During aging, environmental stressors and mutations along with reduced DNA repair cause germ cell aneuploidy and genome instability, which limits fertility and embryo development. Benevolent commensal microbiota and dietary plants secrete indoles, which improve healthspan and reproductive success, suggesting regulation of germ cell quality. Indoles prevent aneuploidy and promote DNA repair and embryo viability, which depends on age and genotoxic stress levels and affects embryo quality across generations. In young animals or with low doses of radiation, indoles promote DNA repair and embryo viability; however, in older animals or with high doses of radiation, indoles promote death of the embryo. These studies suggest indole integrates DNA repair and cell death responses to preclude germ cell aneuploidy and ensure transgenerational genome integrity.

Indoles limit age-dependent intergenerational increases in males and reduces embryo lethality in C. elegans

The incidence of males and embryo lethality in the progeny increase as animals age, an effect attributed to increased oocyte aneuploidy. To evaluate the effects of indole on aneuploidy during normal aging, the incidence of males and dead embryos produced by young [day 1 (DI) to D3] and old (D3 to D5) N2 animals were compared. Whereas indole had no effect in young animals, it reduced male frequencies and embryo lethality in old animals to levels similar to that seen in young animals. [H]im-19 mutants were reported to exhibit a twofold increase in the frequency of males as they age. Whereas indole had no detectable effect on male frequencies in young him- 19 mutants, it reduced the frequency of males in old D3 to D5 animals to a level seen in young animals. Effects of indole on embryo lethality were also assessed following X-irradiation of young and old N2 animals. Animals were irradiated at either DI or D3, and the viability of their embryos were evaluated over the next 2 days. Embryo lethality associated with radiation increased significantly from DI to D3. Whereas indole did not affect the viability of embryos from DI animals, it significantly reduced embryo lethality in D3 animals.

To decipher the genetic pathway mediating the effects of indole during aging, the embryo lethality of mutants in the 9-1-1 pathway [hus-l(op241)], NHEJ pathway [cku-70(tml524)], and MRN pathway [atm-l(gkl86)] grown with or without indole was compared at DI and D3. Unlike the hus-l(op241) and cku-70(tml524) mutants, indole had no effect on age-dependent increases in embryo lethality in the MRN pathway mutant atm-l(gkl86). These data indicate that old animals are more susceptible to damage associated with environmental stressors such as radiation, various mutations (e.g., him-19) have a more pronounced impact on nondisjunction in aged animals compared to younger ones, and indole reduces male frequencies and increases embryo viability in old animals. These data also indicate that indole acts via the MRN pathway to suppress age-dependent increases in embryo lethality. Indoles act via the AhR to regulate nondisjunction in C. elegans

Aryl hydrocarbon receptor (AhR) mediates the effects of indole on healthy aging, raising the possibility that indole also acts via AhR to regulate aneuploidy and embryo lethality. Mutant worms, ahr-l(ia3), display a significantly higher frequency of male progeny compared to N2 animals in both unstressed [0.8% in ahr-l(ia3) versus 0.3% in N2] and heat-stressed [9.4% in ahr- 1 (ia3) versus 3.2% in N2] conditions. Whereas indole reduced male frequencies in N2 animals, it had no effect in ahr-l(ia3) animals. The ahr-l(ia3) animals exhibited higher embryo lethality without stress compared to N2, an effect that was exacerbated by heat stress. In contrast to N2, indole did not abrogate embryo lethality with or without heat stress in ahr-l(ia3) animals. Indole also failed to suppress radiation-induced increases in numbers of males and embryo lethality in ahr-l(ia3) mutants. Indole also did not affect age-dependent increases in the percentage of males and embryo lethality in ahr-l(ia3) mutants. The absence of an indole effect in these various conditions was recapitulated in a second loss-of-function ahr-1 mutant, ahr-l(jul45). Together, these data indicate that indole acts via ahr-1 to limit aneuploidy and promote embryo viability in response to both stressors and aging.

ICA limits X-irradiation-induced DNA damage and promotes DNA repair in fibroblasts and splenocytes

Experiments were performed to determine whether the effects of indole on DDR and aneuploidy is conserved in mammalian somatic and germline cells. Indole-3 -aldehyde (ICA) was used in mammalian experiments. Fibroblasts (3T3) were exposed to X-irradiation in the presence or absence of ICA, and the cytochalaisin B-micronucleus assay was used to quantify cells with unrepaired DNA damage, missegregated chromosomes, or chromosomal breakage. ICA reduced the percentage of cells with one or more micronucleus at all X-irradiation doses tested, despite increased numbers of micronuclei at higher radiation doses.

The micronucleus assay cannot distinguish the effects of X-irradiation on spindles versus chromosomes, both of which could affect chromosome segregation. To directly assess the effects of ICA on single- or double- stranded DNA breaks, alkaline comet assays were performed on X- irradiated 3T3 cells to assess the electrophoretic mobility of nuclear DNA. In a comet assay, cells are embedded in agar and electrophoresed, stained with 4',6-diamidino-2-phenylindole (DAPI), and imaged. Imaged cells resemble a “comet” with a distinct head and tail. The head is composed of intact DNA, while the tail consists of damaged DNA with single-stranded breaks or DSBs or broken pieces of DNA. The extent of DNA liberated from the head into the tail of the comet is directly proportional to the amount of DNA damage.

Treatment with ICA reduced percent tail DNA, the tail moment, and the olive moment, all direct measures of the extent of DNA damage. To confirm that ICA regulated DNA repair, 3T3 cells were X-irradiated and, at various times thereafter, stained with phospho-y-H2A histone family member X (H2AX) monoclonal antibody, which recognizes DSBs. Compared to vehicle- treated cells, ICA had no effect on the number of y-H2AX foci detected per nucleus at the 5-min time point. However, by 20 min, ICA decreased the number of foci compared to vehicle treatment, an effect further enhanced at 5 hours after irradiation. Data from histograms of the frequency of nuclear y-H2AX foci per nucleus indicate that ICA promotes repair of DNA DSBs in mitotic mammalian cells.

Experiments were performed to evaluate the effects of ICA on DDRs in somatic cells during aging in vivo. To do this, 3- or 18-month-old C57B1/6 mice were treated with either vehicle or ICA for 2 weeks, and their splenocytes were isolated, X-irradiated, and subjected to alkaline comet assays 5 or 20 min later. A reduction in comet tail DNA was evident when comparing the 5- and 20-min time points in young animals treated with vehicle, indicative of active repair of DNA damage, and ICA did not provide additional benefit. By contrast, limited repair was evident between the 5- and 20-min time points in old animals treated with vehicle, but ICA reduced comet tail DNA at both time points. These data indicate that splenocytes from young animals retain high DNA damage repair activity, which decreases with age, and ICA restored repair activity in old animals to levels seen in young ones.

ICA acts via AhR and p53 to limit X-irradiation-induced DNA damage in mammals

To determine whether effects of ICA on DDR in mammals depend on AhR and p53, splenocytes were isolated from 20-month-old ahr-/- mice and their age-matched C57B1/6 wildtype counterparts or 2-month-old p53— /— mice and their age-matched C57B1/6 counterparts. Splenocytes were treated ex vivo with ICA, X-irradiated, and subjected to comet assays. Whereas ICA reduced comet size in splenocytes from the C57B1/6 animals, it had no effect on splenocytes derived from ahr-/- or p53— /— animals. Moreover, tail percentages were higher in splenocytes from ahr-/- and p53— /— animals even without radiation compared to those from the C57B1/6 mice, indicating a higher level of baseline damage, lower repair potential, or both. Together, these data indicate that ICA acts via AhR and p53 to facilitate repair of damaged DNA and that these genes mediate protective effects of indoles in vivo.

Commensal E. coli protect mammalian splenocytes from X-irradiation-induced DNA damage

Experiments were performed to determine whether gnotobiotic colonization of the mammalian gut with a commensal bacterial strain producing indoles exerted a DDR akin to that of enteral administration of ICA. To do this, C57B1/6 mice were treated with streptomycin to reduce the microbial diversity and numbers within the intestinal tract and recolonized with either StrpR E. coli KI 2, which produces indole and indole derivatives, or StrpR E. coli K12AtnaA, which does not. The mice maintained the strains for up to 3 months. After 2 months, splenocytes were isolated, X-irradiated, and subjected to comet assays 20 min later. Splenocytes derived from K12-colonized animals exhibited lower percentage tail DNA and reduced tail and olive moments compared to those in splenocytes from K12AtnaA-colonized mice. These data indicate that indoles derived from a commensal E. coli strain limit radiation-induced DNA damage.

ICA limits DNA damage in young mammalian oocytes

Observations in splenocytes suggested that ICA facilitates DNA repair in mitotically dividing mammalian somatic cells. To determine whether ICA likewise limits DNA damage in mammalian germline cells, especially during aging, DDRs were assessed in mammalian oocytes (Fig. 1A). To collect oocytes, 3-month-old CD1 mice were administered either ICA or vehicle for up to 2 weeks, treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) to induce superovulation. Oocytes were then collected from the ampulla, X-irradiated (4-Gy), allowed to recover for 5 min, and then subjected to alkaline comet assays. As shown in Fig. IB, oocytes derived from mice treated with ICA exhibited reduced percentage of tail DNA compared to vehicle controls, indicating that, as with mitotic cells, ICA limited the sensitivity of oocytes to DNA damage from X-irradiation. ICA induces fragmentation and reduces viability of oocytes from aged mammals

To determine whether indole regulates mammalian oocyte quality with aging, the morphology of the oocytes was evaluated following ovulation and subsequent development was monitored following in vitro fertilization (IVF) (Fig. 1A). First, 3-month-old (young) CD1 mice were exposed to ICA or carrier for up to 2 weeks, harvested oocytes from the ampulla 13 hours after superovulation. Their morphology and capacity to form blastocysts following IVF with sperm derived from 3-month-old males was assessed. Young animals yielded large numbers of intact oocytes (approximately 28 per animal) 13 hours after induction of superovulation with hCG (Fig. 1C). Oocytes with abnormal morphology such as fragmentation were not observed (Fig. ID), and, upon IVF, 70% of the oocytes were successfully fertilized, as assessed by the appearance of two polar bodies and two distinct pronuclei 8 to 10 hours later (Fig. IE). Most of the fertilized oocytes developed into blastocyst after 96 hours with only 10% of the embryos exhibiting developmental arrest (Fig. IF). The oocytes derived from mice treated with ICA exhibited similar scores in all these assays indicating the absence of an effect in young mice.

Experiments were performed to determine the effects of indoles on oocytes derived from 8-month-old (old) CD1 female mice. Notably, oocytes from this strain exhibit loss of quality by 6 to 9 months of age. Old CD1 females were administered ICA or vehicle for up to 2 weeks. Upon superovulation, the number of oocytes recovered from older animals was significantly reduced compared to young animals. As with young animals, pretreatment of old mice with ICA was without effect on oocyte yield (Fig. 1C). Thus, ICA did not affect the age-associated decline in the number of oocytes induced to ovulate with hormone treatment. However, oocytes derived from old animals administered ICA exhibited significantly higher rates of fragmentation, compared to rates from vehicle-treated animals (Fig. ID). Moreover, upon IVF, fertilization rates were markedly reduced, and developmental arrest was evident in a higher fraction of fertilized oocytes derived from ICA-treated animals as compared to rates from vehicle-treated animals (Fig. IE and IF).

To determine whether the effects of ICA were specific to CD1 mice or associated with aging per se, its effects were assessed on oocytes derived from young C57B1/6 females, previously treated with ICA or vehicle. Oocytes were allowed to mature in the ampulla for 24 hours instead of 13 hours after superovulation. This protocol, called “postovulatory aging,” induces fragmentation in oocytes and recapitulates the aging of oocytes in the absence of sperm or fertilization. While ICA did not affect young oocytes harvested at 13 hours (Fig. 1C), prior exposure to ICA of oocytes harvested at 24 hours resulted in an increased frequency of oocytes displaying fragmentation or abnormal morphology when compared to vehicle controls (Fig. 1G). Together, these data indicate that ICA was without effect on fragmentation or postfertilization development of oocytes harvested from young animals, However, ICA increased oocyte fragmentation and reduced viability of oocytes subjected to the postovulatory aging protocol. Thus, providing a utility in identifying more desirable oocytes for IVF by separating unfragmented oocytes from fragmented oocytes with reduced viability.

Claims

1. A method of isolating an oocyte or ovum for use in in vitro fertilization comprising contacting a sample comprising oocyte or ovum with indole or derivative providing an indole preserved oocyte or ovum.

2. A method of preserving an oocyte or ovum for use in in vitro fertilization comprising contacting an isolated oocyte or ovum with indole or derivative and storing or culturing the oocyte or ovum providing an indole preserved ovum.

3. The method of claim 2, further comprising contacting the indole preserved oocyte with sperm providing an embryo.

4. The method of claim 2, further comprising implanting the embryo in a uterus of a female subject.

5. A method of purifying an oocyte for use in inducing the formation of ovum comprising: contacting a sample comprising oocytes with indole or derivative providing a indole preserved oocytes; detecting in the sample an oocyte that is abnormal; separating the abnormal oocyte providing a purified preserved oocyte; and contacting the purified preserved oocyte with sperm inducing formation of an ovum.

6. A method for improving the fertility of a female subject comprising administering to a female subject an effective amount of an indole or derivative thereby improving fertility in a female subject.

7. The method of claim 6, wherein the indole or derivative is orally or systemically administered to the female subject.

8. The method of claim 6, wherein the indole or derivative is administered locally to the ovary of the female subject.

9. A method for improving the recovery of ovulated oocytes comprising administering to a female subject an effective amount of an indole or derivative.

10. The method of claim 9, wherein the indole or derivative is orally or systemically administered to the female subject.

11. The method of claim 9, wherein the indole or derivative is administered locally to the ovary of the female subject.

12. The method of claim 9, further comprising obtaining an oocyte from a female subject and forming a fertilized ovum by in vitro fertilization.

13. The method of claim 12, further comprising implanting the fertilized ovum in the female subject wherein the pregnancy outcome of the female subject is improved compared to a control standard.

14. A composition comprising an oocyte or progeny cell and indole or derivative.

15. The composition of claim 14, wherein the progeny cell is an ovum.

16. The composition of claim 14, further comprising leuprolide acetate, gonadotropin releasing horm one-agonists, follitropin beta, urofollitropin, follicle stimulating hormone (FSH)), combination of FSH and LH, luteinizing hormone, human chorionic gonadotropin (hCG), or combinations thereof.

17. The composition of claim 14, further comprising nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinic acid, tryptophan, quinolinic acid, fisetin, quercetin, resveratrol, hydroxytyrosol, pyrroloquinoline quinone, metformin, apigenin, luteolin, berberine, or combinations thereof.

18. The composition of claim 14, which is a cell culture medium, an oocyte collection solution, an oocyte washing solution, an oocyte in vitro maturation medium, an oocyte in vitro fertilization medium, an embryo medium, vitrification solution, cryopreservation solution, or embryo thawing medium.