Compositions and methods for utilizing extracellular vesicles (EVS) to improve fertility in humans and non-human animals

Abstract

Embodiments of the instant disclosure relate to compositions and methods for enhancing fertility in humans and non-human animals. In certain embodiments, the instant disclosure relates to making and using conditioned extracellular vesicles (EVs) from epididymal epithelial cells (EECs). In other embodiments, the conditioned EVs disclosed herein can be created for use in inducing sperm motility and improving fertility in humans and non-human animals. In some embodiments, conditioned EVs can be used to induce sperm motility by at least five percent (5%) compared to controls not exposed to the conditioned EVs created herein. Certain embodiments related to compositions containing conditioned EVs, sperm and a medium.

Description

106549-802378-CU6205H-02-PCT1 COMPOSITIONS AND METHODS FOR UTILIZING EXTRACELLULAR VESICLES (EVS) TO IMPROVE FERTILITY IN HUMANS AND NON-HUMAN ANIMALS PRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 524,799 filed July 3, 2023. This provisional application is incorporated herein in its entirety for all purposes. GOVERNMENT FUNDING

[0002] These inventions were made with government support under Grant No. MH108286 from the National Institute of Health (NIH). The government has certain rights in these inventions. FIELD

[0003] Embodiments of the instant disclosure relate to compositions and methods for enhancing fertility in humans and non-human animals. In certain embodiments, the instant disclosure relates to making and using conditioned extracellular vesicles (EVs) from epididymal epithelial cells (EECs). In other embodiments, the conditioned EVs disclosed herein can be created for use in inducing sperm motility and improving fertility in humans and non-human animals. In some embodiments, conditioned EVs can be used to induce sperm motility by at least five percent (5%) compared to controls not exposed to the conditioned EVs created herein. Other embodiments relate to compositions containing conditioned EVs, sperm and a medium. BACKGROUND

[0004] Issues of fertility are a problem in both human and non-human animals. The current state of the art for improving human and non-human fertility can frequently involve artificial insemination or in vitro fertilization. The ability of non-human animals to reproduce efficiently is an integral component of animal agriculture and successful passage of superior traits from one animal to an offspring. Therefore, reliable and efficient fertilization is required to reproduce effectively and efficiently. Infertility is a problem in all non-human animal production systems, including aquaculture species. Reproductive failure is one of the most significant factors that limit the productivity of non-human animal production systems and lead to multimillions of dollars in unsuccessful fertilizations resulting in significant losses annually in time, resources, and money. Therefore, a need exists for improved and reliable methods for fertilization of human and non-human animals. 1 95743809.1106549-802378-CU6205H-02-PCT1 SUMMARY

[0005] Embodiments of the instant disclosure relate to compositions and methods for enhancing fertility in humans and non-human animals. In certain embodiments, the instant disclosure relates to making and using conditioned extracellular vesicles (EVs) from epididymal epithelial cells (EECs). In some embodiments, the conditioned EVs disclosed herein can be created for use in inducing sperm motility and improving fertility in humans and non-human animals. In some embodiments, conditioned EVs can be used to induce sperm motility by at least five percent (5%) or more, compared to controls not exposed to the conditioned EVs created herein. Certain embodiments related to compositions containing conditioned EVs, sperm and a medium.

[0006] In certain embodiments and further to paragraph

[0005] above, compositions disclosed herein can include conditioned extracellular vesicles (EVs); at least one sperm, and a medium. In some embodiments, the conditioned EVs are derived from epididymal epithelial cells (EECs) exposed to at least one glucocorticoid for a predetermined period. In certain embodiments, the at least one glucocorticoid includes, but is not limited to, at least one of cortisone, cortisol, corticosterone, or corticosteroid type agent or the like, hydrocortisone, prednisone, dexamethasone, or other known glucocorticoid or a combination thereof. In other embodiments, the conditioned EVs can be harvested from EECs after exposure to at least one glucocorticoid for about 1 hour to about 1 week (e.g., 72 hours of incubation) and then incubated for at least one hour to about 2 weeks (e.g., 6 days of incubation) without the at least one glucocorticoid.

[0007] In certain embodiments and further to paragraph

[0005] -

[0006] above, compositions disclosed herein can include conditioned extracellular vesicles (EVs) where the conditioned EVs have increased production of, or expression of R-spondin family proteins (RSPO family, e.g., RSPO1-4) compared to un-conditioned EVs. In other embodiments, conditioned EVs disclosed herein can have increased production of, or expression of at least one of Thbs1, Thbs3, Thbs4, ATP5a, ATP5b, as well as other ATP synthase proteins compared to un-conditioned EVs. In accordance with these embodiments, increased levels, concentrations, or expression of these R- spondin family proteins and / or Thbs1, Thbs3, Thbs4, ATP5a, ATP5b, as well as other ATP synthase proteins can be about 1.1 to about 1.9-fold or more, or about 1.1 to about 1.8-fold or more, higher concentration or levels or expression compared to un-conditioned EVs. 2 95743809.1106549-802378-CU6205H-02-PCT1

[0008] In some embodiments and further to paragraph

[0005] -

[0007] above, conditioned EVs can have increased mitochondrial ATP production compared to un-conditioned EVs. In yet other embodiments, the conditioned EVs can have increased conditioned EV-sperm fusion compared to unconditioned EVs. In other embodiments, increased expression of proteins in conditioned EVs can include increased production of, or expression of one or more of Stx7 and Rab35 or other known protein or proteins to increase fusion of EVs with sperm to improve sperm motility. In accordance with these embodiments, increased levels, concentrations, or expression thereof can be about 1.1 to about 1.3-fold or more, or about 1.1 to about 1.2-fold or more, higher concentration or levels and / or expression compared to un-conditioned EVs. In some embodiments, conditioned EVs can be produced and isolated from any cell culture media. In other embodiments, conditioned EVs can be produced and then isolated from a Iscove's Modified Delbecco's Medium (IMDM), DMEM, or the like in the presence or absence of serum (e.g., fetal bovine or other serum at about 1.0 to about 20.0 % w / v). In certain embodiments, the conditioned EVs disclosed herein can display two or more of these characteristics compared to EVs not exposed to the conditions disclosed herein (e.g., one or more glucocorticoid and at least one post exposure wash and incubation).

[0009] In certain embodiments and further to paragraphs

[0005] -

[0008] above, the compositions disclosed herein can include at least one additional agent including, but not limited to, an agent to enhance sperm motility or preserve sperm viability.

[0010] In certain embodiments and further to paragraphs

[0005] -

[0009] above, compositions disclosed herein can include sperm from humans. In other embodiments, compositions disclosed herein can include sperm from non-human animals such as non-human food animals, fish and / or birds or any animal bred using artificial insemination processes. In yet other embodiments, compositions disclosed herein can include livestock or companion animal or pet sperm. Other embodiments concern compositions including bird or avian sperm (e.g., chicken, turkeys, or other birds) or aquatic species sperm (e.g., farm grown fish or other aquatic species).

[0011] In some embodiments and further to paragraphs

[0005] -

[0010] above, methods for improving sperm motility are disclosed. In accordance with these embodiment, methods can include, but are not limited to, obtaining conditioned EVs from EECs; and introducing the conditioned EVs to sperm in a composition comprising a medium. In other embodiments conditioned EVs include, but are not limited to, EVs exposed to at least one glucocorticoid for 3 95743809.1106549-802378-CU6205H-02-PCT1 about 1 hour to about 1 week or more as appropriate (e.g., 36 to about 96 hours) and then incubated for at least one hour to about 2 weeks (e.g., about 1 day to about 8 days of incubation) without the at least one glucocorticoid. In certain embodiments, the at least one glucocorticoid can be removed, and the cells washed one or more time and placed in glucocorticoid-free media for the post exposure period of about one hour to about 2 weeks. It was discovered that this post incubation period without the at least one glucocorticoid is important in the production of the conditioned EVs. In other embodiments, methods can include introducing at least one additional agent to the composition to improve sperm motility, improve sperm viability, improve fertilization outcome or combinations thereof.

[0012] In other embodiments and further to paragraphs

[0005] -

[0011] above, in vitro methods for improving fertility in a subject are disclosed. In some embodiments, methods include, but are not limited to, providing sperm exposed to conditioned EVs of a composition disclosed herein to an egg of a subject in an in vitro environment to improve fertilization and / or improve chances of fertilization of the egg. In other embodiments, the egg exposed to a sperm provided conditioned EVs disclosed herein can further include introducing the fertilized egg to a subject for maturation or for further incubation and growth of an embryo by artificial means or natural means.

[0013] Certain embodiments and further to paragraphs

[0005] -

[0012] above, concern kits for use in increasing sperm motility and improving fertility in a subject. In some embodiments, kits can include conditioned EVs and at least one container. In other embodiments, kits can further include media for stabilizing the conditioned EVs alone or in combination with sperm. In other embodiments, kits can include compositions or aliquots of compositions of conditioned EVs, sperm and at least one storage media for freezing and / or storing compositions for later use. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIGS.1A-1P illustrate in 1A a schematic of cells under certain conditions is represented; in 1B a representative graph illustrating features of cells over time with and without glucocorticoid exposure; 1C-1G illustrate plots of treated versus control measurements and 4 95743809.1106549-802378-CU6205H-02-PCT1 comparisons of various features; 1H-1K illustrates representative photographic representations of electron micrographs of cells with and without exposure to a glucocorticoid; 1L-1N illustrate control versus treated cells and measurement of various parameters as indicators; 1O illustrates a time course of various treatments to control and glucocorticoid exposed cells and changes in various parameters; and 1P illustrates a graph with media supplementation in control and glucocorticoid treated cells and comparison of the treated and untreated cells under various conditions of certain embodiments disclosed herein.

[0016] FIGS.2A-2H illustrate in 2A a schematic illustration of a proposed underlying mechanism for producing conditioned EVs of use herein such as increasing sperm motility; 2B 2B is a representative plot over time analyzing the cells’ metabolic response to substrates known to be involved with the cells’ mitochondrial electron transport chain (involved in mechanisms important for producing conditioned EVs); 2C-2F illustrate plots comparing treated and untreated control samples regarding parameters as indicated; 2G illustrates a plot of enzymatic activity measured in treated versus control samples; and 2H illustrates a photographic image of fluorescent representation of Western blot analysis expression of various target proteins and a bar graph of ratios of the same target proteins of certain embodiments disclosed herein.

[0017] FIGS.3A-3H illustrate in 3A, a schematic diagram of a cell treatment process and further incubation without treatment of target cells for EV production; 3B illustrates control versus treated gene expression of certain embodiments disclosed herein; 3C represents is a schematic drawing illustrating a representative induction of a particular protein and it's binding abilities that can be involved in pathways induced by treatments disclosed herein; 3D illustrates region percentages correlating with binding of the particular protein in 3C; 3E illustrates binding site distribution of the particular protein in 3C; 3F illustrates changes in treated versus control vehicle treated cells expression of the particular protein in 3C; 3G illustrates additional parameters of expression; and 3H illustrates gene set enrichment of various genes that align with induction of the target protein and its binding in certain embodiments disclosed herein.

[0018] FIGS.4A-4I illustrate in 4A, a schematic diagram of a cell treatment process and further incubation without treatment with a change in media at a particular day of incubation of target cells for EV production; 4B illustrates EV concentration distribution with and without glucocorticoid treatment compared to control vehicle treated cells and a negative control; 4C illustrates area under the curve representing potential changes in particle distribution; 4D 5 95743809.1106549-802378-CU6205H-02-PCT1 illustrates EV particle size distribution in glucocorticoid and control vehicle treated cells after incubation as in 4A; 4E represents a schematic diagram of a cell treatment after the process of treatment and further incubation with sperm and assessment of whole cell respirometry under the 2 conditions; 4F-4I illustrates exemplary plots of parameters measured in sperm under treated and control treated EV conditions where the EVs have been mixed with sperm of certain embodiments disclosed herein.

[0019] FIGS.5A-5H represent in 5A a schematic diagram of a cell treatment after the process of treatment and further incubation with sperm and assessment of migration and motility; 5B-5H illustrate motility parameters measured in sperm mixed with treated and control vehicle treated and harvested EVs (conditioned or unconditioned control EVs) under test conditions of certain embodiments disclosed herein.

[0020] FIGS.6A-6C represent bar graphs of parameters of control vehicle versus treated cells’ mitochondria regarding area (6A), perimeter (6B) and circularity (6C) of certain embodiments disclosed herein.

[0021] FIGS.7A-7I represent in 7A, a schematic of cells under certain conditions disclosed herein. Expression relative to non-transfected EEC expression. (7B). Baseline mitochondrial oxygen consumption rate of cells exposed to the treatment represented in 7A (7C). Schematic of the EEC in vitro treatment paradigm (7D). Baseline mitochondrial oxygen consumption rate exposed to the paradigm in (7E, 7D). Schematic of the EEC in vitro treatment paradigm (7F). Baseline mitochondrial oxygen consumption rate of cells exposed to the paradigm (7G) compared to a control (7F). GR(glucocorticoid receptor) protein band volume relative to H3 protein band volume from cells’ nuclear fraction. (7H). GR protein band volume relative to Atp- 5a protein band volume from cells’ mitochondrial fraction, (7I).

[0022] FIGS. 8A-8B represent a peak aligning with a reference protein demonstrating increased target protein binding at the promoter region in cells with prior treatment (8A). The reference gene is highlighted in blue, while the promoter region is demarcated below the figure. Peaks are presented in rows and samples are presented in columns (8B).

[0023] FIGS. 9A-9D illustrate in 9A, a schematic of the cell-EV incubation protocol. Decreased cell mitochondrial oxygen consumption rate following incubation with conditioned- EVs but not vehicle-EVs, (9B). Decreased ATP Production rate following conditioned-EV 6 95743809.1106549-802378-CU6205H-02-PCT1 incubation, (9C). Decreased cell mitochondrial oxygen consumption rate following incubation with conditioned-EVs but not vehicle-EVs, (9D). DEFINITIONS

[0024] Terms, unless defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0025] Unless otherwise indicated, all numbers expressing quantities of agents and / or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that can vary from about 10% to about 15% plus and / or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

[0026] As used herein, “individual”, “subject”, “host”, and “recipient” can be used herein and refer to any subject for whom improved fertilization or improved sperm motility is desired, for example, humans, or non-human animals, other mammals, horses or other companion animals or pets, livestock (e.g., sheep, cattle, buffalo, alpacas, pigs, rabbits, etc.) birds or aquatic subjects such as fish. DETAILED DESCRIPTION OF THE INVENTION

[0027] In the following sections, certain exemplary compositions and methods are described to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0028] Issues of fertility are a problem in both human and non-human animals. The current state of the art for human fertility can frequently involve artificial insemination or in vitro fertilization both of which involve rinsing or washing of sperm. It is considered that this process removes beneficial agents. Embodiments disclosed herein consider agents that improve fertility by providing compositions to improve sperm motility and stability. Embodiments disclosed herein provide unique cellular signals provided by conditioned EVs produced from EV- 7 95743809.1106549-802378-CU6205H-02-PCT1 producing cells that have treated with glucocorticoids or similar agents and then incubated without glucocorticoids for a predetermined period. In certain embodiments, synthetic or conditioned EVs disclosed herein expressing specific proteins and RNAs deliver signals to increase sperm motility and enhance implantation rates for improved fertility in a subject. In some embodiments, conditioned EVs disclosed herein represent modified EVs that mimic at least some stress conditions or stressed state as observed post-exposure to the at least one glucocorticoid.

[0029] In other embodiments, with respect to animal husbandry and ranching, non-human animals or food animals can be targeted with approaches for improving fertility by compositions and methods disclosed herein, in addition to improving human fertility. In accordance with these embodiments, livestock and other food animal production can incur significant losses due to reduced fertility. The ability of non-human animals or food animals to reproduce efficiently is an integral component of livestock, companion animal, and other animal production and for successful passage of superior traits from one animal to an offspring. Infertility is a problem in all non-human animal production systems and AI is used to improve these outcomes. Reproductive failure is one of the most significant factors that limit the productivity of non- human animal production systems and result in multimillions of dollars in unsuccessful fertilizations resulting in significant losses annually.

[0030] One major challenge facing many producers of livestock and other animals using artificial insemination (AI), is finding practical, cost-effective ways to improve reproductive performance without compromising the quality and soundness of the resulting progeny or production of safe, high quality animal products. In certain embodiments disclosed herein, to improve animal production, incubation of sperm with conditioned EVs disclosed herein can increase rates of pregnancy, enhancing fertilization efficiency from all artificial insemination (AI) methods. In other embodiments, unlike many other methods used in developed countries such as transgenics or cloning, incubation of sperm with conditioned EVs disclosed herein can easily be applied and incubated with sperm samples without expensive high tech equipment improving animal and food animal production in developed and in developing countries in need. In developed countries and around the world, AI is the predominant assisted reproduction method used in animal food production of beef, pork, lamb, dairy, fish, and poultry production. 8 95743809.1106549-802378-CU6205H-02-PCT1

[0031] It is known that chronic stress and traumatic experiences are associated with neuropsychiatric and metabolic diseases including depression, post-traumatic stress disorder, obesity, and metabolic syndrome. It has been observed that prolonged effects of chronic stress on health outcomes occur long after the stressor ends, indicating a period of cellular programming for long-term adaptation to stress exposure. It has also been observed that long term adaptation following chronic stress induces a new allostatic state, or set point, over time.

[0032] In certain embodiments and further to paragraphs

[0027] -

[0031] above, it is known that in males, somatic epididymal epithelial cells (EECs) produce biological nanoparticles, extracellular vesicles (EVs) that convey signals essential for sperm maturation. Disclosed herein are systems for harnessing optimum processes of conditioned EVs for use in conveying certain traits to exposed sperm samples for improved fertility. In certain embodiments, lasting effects of cells producing conditioned EV cargo can influence downstream physiology and function of targeted cell types (e.g., sperm cells).

[0033] In other embodiments and further to paragraphs

[0027] -

[0032] above, low affinity glucocorticoid receptors (GRs) are sensitive to timing and duration of glucocorticoid signaling relative to the daily changes that occur with the diurnal rhythm and it was proposed that exposure of cells to glucocorticoids could engage this process to produce conditioned EVs. GR is a key regulator of cellular processes important for mitochondrial and transcriptional functions, placing it in a nodal point in most cells for partitioning resources and energy. In certain embodiments, recalibration of cells for certain allostatic set points following chronic stress such as glucocorticoid exposure is mediated at least in part at the level of mitochondrial function and nuclear regulation of cellular energy requirements, altering cellular metabolic states, and resulting in changes to cellular processes and communication including EVs required for sperm function. Further, sperm motility is the ability of sperm to move efficiently in order to fertilize an available egg. It is noted that motility is important in fertility in general and in AI it is important to allow the sperm to reach and fertilize the egg. Poor sperm motility is a significant cause of male factor infertility in humans and non-human animals such as food animals.

[0034] Embodiments of the instant disclosure and further to paragraphs

[0027] -

[0033] above, compositions and methods relate to enhancing fertility in humans and in non-human animals. In certain embodiments, the instant disclosure relates to making and using conditioned extracellular vesicles (EVs) from cells (e.g., epididymal epithelial cells (EECs)). In some 9 95743809.1106549-802378-CU6205H-02-PCT1 embodiments, the conditioned EVs disclosed herein can be created for use in inducing sperm motility and other traits for example, for improving fertility in humans and non-human animals. In some embodiments, conditioned EVs can be used to induce sperm motility by at least five percent (5%), up to ten percent (10%), up to fifteen (15%), up to twenty percent (20%) or any percentage in between or more than 20%, compared to sperm or control sperm not exposed to the conditioned EVs created herein. Certain embodiments disclosed herein concern compositions including, but not limited to, conditioned EVs, sperm and a medium for use in enhancing fertility.

[0035] In certain embodiments and further to paragraphs

[0027] -

[0034] above, compositions disclosed herein can include conditioned extracellular vesicles (EVs); at least one sperm, and a medium. In accordance with these embodiments, the conditioned EVs are derived from epididymal epithelial cells (EECs) exposed to at least one glucocorticoid or similar agent for a pre-determined period. In certain embodiments, the at least one glucocorticoid includes, but is not limited to, at least one of cortisone, cortisol, corticosterone or corticosteroid type agent or the like, hydrocortisone, prednisone, dexamethasone, a combination thereof or other glucocorticoid capable of inducing a stressed-state in the EECs. In other embodiments, the conditioned EVs can be harvested from cells (e.g., EECs) or harvested from media of conditioned cells after exposure to at least one glucocorticoid or similar agent for about 1 hour to about 2 weeks, or about 6 hours to about 1 week, or about 12 hours to about one week, or about 18 hours to about 1 week, or about 24 hours to about one week, or about 30 hours to about one week, or about 36 hours to about one week, or about 42 hours to about one week, or about 48 hours to about one week, or about 54 hours to about one week, or about 60 hours to about 1 week, or about 66 hours to about one week, to about 72 hours to about 1 week, or about 78 hours to about one week, or about 84 hours to about 1 week or more. In some embodiments, the conditioned EVs can be harvested from cells (e.g., EECs) or harvested from media of conditioned cells (e.g., EECs) after exposure to at least one glucocorticoid or similar agent for about 12 hours to about 4 days. In some embodiments, the cells (e.g., EECs) can be harvested, washed and / or placed into a glucocorticoid-free or similar-free media and incubated for at least one hour to about 2 weeks or about 6 hours to about 2 weeks, or about 12 hours to about 2 weeks, or about 18 hours to about 2 weeks, or about 24 hours to about 2 weeks, or about 30 hours to about 2 weeks, or about 36 hours to about 2 weeks, or about 42 hours to about 2 weeks, 10 95743809.1106549-802378-CU6205H-02-PCT1 or about 48 hours to about 2 weeks, or about 54 hours to about 2 weeks, or about 60 hours to about 2 weeks, or about 66 hours to about 2 weeks, to about 72 hours to about 2 weeks, or about 78 hours to about 2 weeks, or about 84 hours to about 2 weeks or more without or in absence of the at least one glucocorticoid or similar agent. In accordance with these embodiments, the cells (e.g., EECs) can be harvested, optionally washed and / or placed into a glucocorticoid-free replacement media and incubated for at least about 12 hours to about 1.5 weeks without the at least one glucocorticoid or equivalent agent to produce conditioned EVs and extrude or excrete EVs in the replacement media; for example, for harvesting and use. In certain embodiments, replacement media can be collected to isolate the conditioned EVs or the conditioned EVs can be harvested by methods known in the art. In some embodiments, conditioned EVs can harvested from media of the post-treated EECs and stored or used immediately to improve sperm motility. In some embodiments, EVs can be isolated from any suitable media known in the art and can include but is not limited to serum supplements.

[0036] In some embodiments, and further to paragraphs

[0027] -

[0035] above, EVs can be isolated and / or at least partially purified. In some embodiments, further, after harvesting conditioned media, conditioned EVs can be centrifuged to obtain EVs. In other embodiments, the supernatant can be again centrifuged and as needed ultracentrifuged. In accordance with these embodiments, EVs are pelleted and the EVs can be resuspended in a suitable buffer. In certain embodiments, resuspended conditioned EVs disclosed herein can be stored (e.g., frozen). In yet other embodiments, resuspended conditioned EVs can be placed in media before or after freezing and combined with sperm (e.g., 1x107to about 1x1010or other suitable number of sperm) and further incubated for a pre-determined amount of time. In certain embodiments, the conditioned EVs and sperm can be incubated for about 1 minute up to several hours or a day as appropriate. In certain embodiments, sperm can be incubated with conditioned EVs disclosed herein for about 1 minute up to one day, or about 1 minute up to several hours, or about 5 minutes to about 12 hours, or about 10 minutes to about 6 hours, or about 10 minutes to about 2 hours, or about 20 minutes to about 1.5 hours, or about 30 minutes to about one hour. In some embodiments, the EVs remain with the sperm for further use in a fertilization process. In other embodiments, post conditioned EV sperm can be harvested without EVs for further use in fertilization process. In other embodiments, the conditioned EVs and sperm can be combined in a media suitable for freezing and storing for later use. 11 95743809.1106549-802378-CU6205H-02-PCT1

[0037] In certain embodiments and further to paragraph

[0027] -

[0036] above, compositions disclosed herein can include conditioned extracellular vesicles (EVs) wherein the conditioned EVs have increased expression of R-spondin family proteins (RSPO family) compared to un- conditioned EVs not harvested from cells exposed to glucocorticoids or similar agent. In some embodiments, the conditioned EVs can have increased mitochondrial ATP production compared to un-conditioned EVs. In other embodiments, conditioned EVs disclosed herein can have increased production of, and / or expression of at least one of Thbs1, Thbs3, Thbs4, ATP5a, ATP5b, as well as other ATP synthase proteins compared to un-conditioned EVs. In yet other embodiments, combinations of these effects can be observed in conditioned EVs compared to un-conditioned EVS. In accordance with these embodiments, increased levels, concentrations or expression can be about 1.1 to about 1.9 or more, or about 1.1 to about 1.8-fold, or more, higher concentration or levels or expression compared to un-conditioned EVs. In some embodiments, ATP5a and ATP5b which are components of the ATP synthase complex of the mitochondrial electron transport chain levels or concentrations can be increased by about 1.4 to about 1.8 or higher fold in conditioned EVs.

[0038] In yet other embodiments and further to paragraph

[0027] -

[0037] above, the conditioned EVs can have increased conditioned EV-sperm fusion compared to unconditioned EVs. In certain embodiments, particular genes or proteins are upregulated or down regulated in conditioned EVs that then affect sperm downstream due to contact / fusion of EVs to sperm. In certain embodiments, the conditioned EVs can display two or more of these characteristics compared to EVs produced from cells not exposed to one or more glucocorticoids or similar agent disclosed herein. In some embodiments, the conditioned EVs can display two or more of these characteristics compared to EVs produced from cells not incubated for the times contemplated herein after exposure to the one or more glucocorticoid or similar agent contemplated herein. In some embodiments, conditioned EVs can be reduced in size compared to unconditioned EVs. In other embodiments, conditioned EVs in the presence of sperm induce respiratory mitochondrial changes where respiration can be induced leading to increased motility of sperm. In certain embodiments, the compositions disclosed herein can include at least one additional agent comprising an agent to enhance sperm motility or preserve sperm viability. 12 95743809.1106549-802378-CU6205H-02-PCT1

[0039] In some embodiments and further to paragraph

[0027] -

[0038] above, increased expression of proteins in conditioned EVs can include increased production of, or expression of one or more of Stx7 and Rab35 or the like, or other known protein or proteins to increase fusion of conditioned EVs disclosed herein with sperm to, for example, improve sperm motility. In some embodiments, conditions EVs can be produced and isolated from any cell culture media. In other embodiments, conditions EVs can be produced and isolated from a Iscove's Modified Delbecco's Medium (IMDM), DMEM, or the like in the presence or absence of serum (e.g., fetal bovine or other serum at about 1.0 to about 20.0 % w / v).

[0040] In certain embodiments and further to paragraphs

[0027] -

[0039] above, compositions disclosed herein can include sperm from humans. In other embodiments, compositions disclosed herein can include sperm from non-human animals such as food animals. In yet other embodiments, compositions disclosed herein can include sperm from livestock or companion animals or pets. Other embodiments concern compositions including sperm from birds (e.g., chicken, turkeys, or other birds) or aquatic species sperm (e.g., farm grown fish or other aquatic species). In accordance with these embodiments, these sperm can be introduced to, combined with, and / or mixed with conditioned EVs disclosed herein to improve male fertilization characteristics and improve fertilization outcomes by increasing sperm motility, for example. In certain embodiments, conditioned EVs improve motility in sperm that are already mobile in a targeted sperm population and improve distance and rate of motility of the already mobile sperm in the targeted sperm population for improved sperm performance and fertility outcomes.

[0041] In certain embodiments and further to paragraphs

[0027] -

[0040] above, compositions and methods for improving sperm motility are disclosed. In accordance with these embodiments, methods can include, but are not limited to, obtaining conditioned EVs from cells (e.g., EECs); and introducing the conditioned EVs to sperm in a composition comprising a medium. In other embodiments conditioned EVs include, but are not limited to, EVs obtained from cells exposed to at least one glucocorticoid for about 1 hour to about 1 week and then incubated for at least one hour to about 2 weeks without the at least one glucocorticoid; optionally after several media changes. In other embodiments, methods can include introducing at least one additional agent to the composition before, during, or after combining conditioned EVs with sperm to improve sperm motility, improve sperm viability, improve fertilization outcome or a combination thereof. 13 95743809.1106549-802378-CU6205H-02-PCT1

[0042] In other embodiments and further to paragraphs

[0027] -

[0041] above, in vitro methods for improving fertility in a subject are disclosed. In some embodiments, methods include, but are not limited to, providing sperm exposed to conditioned EVs of a composition disclosed herein to an egg of a subject in an in vitro environment to improve fertilization of the egg. In other embodiments, the egg exposed to a sperm exposed to conditioned EVs disclosed herein can further include introducing the fertilized egg to a subject for further maturation and development or incubating the fertilized egg in an artificial environment. In accordance with these embodiments, the conditioned EVs improve sperm characteristics for enhancing chances of successful fertilization. In some embodiments, these compositions and methods can be used when analysis of a subject’s sperm indicates a need for improving sperm characteristics such as increasing sperm motility for increased chances of successful fertilization. In other embodiments, conditioned EVs can be provided to a sperm population as a general practice to improve chances of fertilization through AI in a targeted animal, bird or fish population, or in a human, without adverse effect on the sperm population.

[0043] Certain embodiments and further to paragraphs

[0027] -

[0042] above, concern kits for use in increasing sperm motility or other characteristics and improving fertility in a subject or subjects through AI fertilization processes. In some embodiments, kits can include conditioned EVs and at least one container. In other embodiments, kits can further include media for stabilizing the conditioned EVs alone or in combination with sperm. In other embodiments, kits can include compositions or aliquots of compositions of EVs, sperm and at least one storage media for freezing and / or storing compositions for later use. In other embodiments, kits can include EECs and compositions for generating conditioned EVs of use herein. In yet other embodiments, kits can include instructions or directions for any kit contemplated herein. It is contemplated that these compositions and methods can be used to enhance fertilization in humans or non-human animal such as food animals, or other animal as appropriate undergoing AI processes in order to save time and money for improved and more predictable fertility leading to improved and predictable reproduction.

[0044] In some embodiments and further to paragraphs

[0027] -

[0043] above, kits can include components for generating conditioned EVs. In some embodiments, the kits can include at least one container appropriate for storing components of a kit disclosed herein or for storing 14 95743809.1106549-802378-CU6205H-02-PCT1 and or dispersing cells or EVs produced from cells or sperm exposed to EVs produced by compositions and methods disclosed herein.

[0045] In certain embodiments and further to paragraphs

[0027] -

[0044] above, a subject contemplated herein is a human subject or other non-human subject such as a food animal where compositions and methods disclosed herein can be used to improve fertility. In accordance with these embodiments, a human subject can include a young adult, adult or older adult.

[0046] In some embodiments and further to paragraphs

[0027] -

[0045] above, formulations suitable for producing conditioned EVs can include a media for optimally growing EECs or equivalent cell for producing EVs. In accordance with some embodiments herein, aqueous solutions can be suitably buffered (e.g., a pH of from about 3.0 to about 9.0) or physiological pH for optimum conditions. Other conditions and media concern optimum conditions for maintaining conditioned EVs alone or in combination with sperm.

[0047] In certain embodiment and further to paragraphs

[0027] -

[0046] above, sperm exposed to EVs produced by compositions and methods disclosed herein can be stored for later use. In accordance with these embodiments, these sperm can be aliquoted into vials and stored by at refrigeration temperatures or frozen such as quick frozen or frozen by compositions and methods known in the art.

[0048] In some embodiments, kits can include instructions for use in accordance with any of the methods described herein. The included instructions can include descriptions of obtaining optimum conditions for improving sperm characteristics and timing thereof.

[0049] In certain embodiments and further to paragraphs

[0027] -

[0048] above, kits disclosed herein include suitable packaging. Suitable packaging includes, but is not limited to, syringes, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. EXAMPLES

[0050] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made to certain examples or some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. 15 95743809.1106549-802378-CU6205H-02-PCT1 Example 1 Cellular allostatic set point is maintained by decreases in basal cellular energy requirements and ATP production rate in EECs with prior stress

[0051] In one exemplary method, to establish that chronic stress confers lasting changes in cellular energy requirements in a population of epididymal epithelial cells (EECs), mitochondrial respiration was analyzed using an in vitro model of chronic stress. Briefly, DC2 distal caput EECs were exposed to corticosterone for 72 hours, and oxygen consumption rate was measured in the Mito Stress Test 6 days later (day 9) to assess lasting effects of prior stress exposure rather than acute metabolic effects of glucocorticoid exposure (FIGS. 1A, 1B). Whole cell respirometry revealed EECs with prior corticosterone exposure have decreased basal mitochondrial respiration (t(21) = 7.539, p <0.0001) (FIG.1C), spare respiratory capacity (t(21) = 4.236, p = 0.0004) (FIG. 1D), total ATP production rate (t(21) = 8.406, p <0.0001) (FIG. 1E), and oxidative ATP production rate (t(21) = 8.471, p <0.0001) (FIG. 1F). However, there was little to no change in glycolytic ATP production rate (t(21) = 1.687, p = 0.8649) (FIG.1G). This oxidative respiratory phenotype was conserved when limiting glycolytic substrates glutamine and pyruvate in the respirometry media (FStress (1, 43) = 74.08, p < 0.0001; FSubstrate (1, 43) = 57.91, p < 0.0001; FStress*Substrate(2, 43) = 1.765, p = 0.1834) (FIGS. 1O, 1P). Transmission electron microscopy (TEM) was utilized to confirm mitochondrial ultrastructure reflected changes observed by whole cell respirometry. TEM micrographs, represented by images in FIGS.1 H-1K, revealed that prior corticosterone exposure increased the proportion of EEC mitochondria with orthodox ultrastructure (Χ2(1, 500) = 6.406, p = 0.01) (FIG. 1M) resulting in a decreased mean mitochondrial gray value (t(557) = 6.566, p<0.0001) (FIG.1N), and ultrastructure score (t(498) = 2.542, p = 0.01) (FIG. 1L) calculated by assigning orthodox mitochondria a score of 1 and condensed mitochondria a score of 2. There were no observed differences in mitochondrial area, perimeter, or circularity (FIGS.6A-6C).

[0052] Regulation of mitochondrial allostatic changes by glucocorticoid receptor (GR) following stress were first assessed by evaluating the compartmentalization of GR in EECs with prior corticosterone exposure. GR protein was found to be decreased in the nuclear fraction (t(4) = 3.3994, p= 0.0162) (FIG.7H) and increased in the mitochondrial fraction at day 9 (t(6) = 2.218, p = 0.0342) (FIG.7I). To examine the role of GR in regulating basal mitochondrial respiration, short hairpin RNA (shRNA) were expressed to reduce GR expression (F(2,15) = 8.092, p = 16 95743809.1106549-802378-CU6205H-02-PCT1 0.0041; with the Tukey post-hoc test between empty vector and reduced GR, p = 0.0037, and non- targeted shRNA and reduced GR, p = 0.0379) (FIGS.7A, 7B) and measured oxygen consumption rate during the Mito Stress Test. While the previously observed hypo-respiratory phenotype was conserved in empty vector and non-targeted shRNA groups (FStress(1, 60) = 61.23, p < 0.0001; FGR expression(2, 60) = 275.1, p < 0.0001; FStress*GR expression(2, 60) = 23.38, p < 0.0001), reducing GR expression eliminated the difference in basal mitochondrial respiration observed in EECs with prior corticosterone exposure (Tukey post-hoc test between vehicle and corticosterone EECs with reduced GR expression, p = 0.9997) (FIG.7C). Importantly, respiration was reduced in control EECs with reduced GR expression compared to the empty vector and non-targeted shRNA groups (Tukey post-hoc test between empty vector vehicle and prior corticosterone EECs, p < 0.0001, and non-targeted shRNA vehicle and prior corticosterone EECs, p = 0.0005) (FIG.7C).

[0053] To review the role of GR as a regulator of lasting mitochondrial allostatic changes, EECs were exposed to a GR antagonist RU-486 about 72 hours after the corticosterone exposure was ceased (FIG. 7D). No difference was observed in the mitochondrial respiration of RU-486 exposed EECs with prior corticosterone exposure compared to vehicle control (FStress(1, 64) = 35.92, p < 0.0001; FRU-486 exposure(1, 64) = 21.20, p < 0.0001; FStress*RU-486 exposure(1, 64) = 23.38, p = 0.0003; with the Tukey post-hoc test between RU-486 / Vehicle EECs and RU- 486 / Corticosterone EECs at p = 0.4024) (FIG.7E). It is noted that the hyporespiratory phenotype was conserved in the control comparison (Tukey post-hoc test between Control / Vehicle EECs and Control / Corticosterone EECs at p < 0.0001) (FIG.7E). In contrast, when EECs were exposed to RU-486 immediately after glucocorticoid( e.g., corticosterone) exposure, prior exposure again reduced day 9 mitochondrial respiration (FStress(1, 66) = 102.7, p < 0.0001; FRU-486 exposure(1, 66) = 11.09, p = 0.0014; FStress*RU-486 exposure(1, 66) = 9.160, p = 0.0035; with Tukey post-hoc test between RU-486 / VehicleEECs at p < 0.0001) (FIG.7F, 7G). Example 2 Decreases in mitochondrial respiration in EECs with prior stress exposure are driven by mitochondrial complex I

[0054] In another exemplary method, to determine oxidative mitochondrial mechanism regulating hypo-respiration following stress, change in respiration (dOCR) was examined after injecting substrates supplying NADH or FADH to the two-electron transport chain entry enzymes, mitochondrial complex I and II (schematic diagram represented in FIG.2A, representative graph 17 95743809.1106549-802378-CU6205H-02-PCT1 2B). dOCR was decreased in EECs with prior corticosterone exposure following injections of glutamate and malate (t(15) = 5.770, p < 0.0001), and pyruvate and malate (t(16) = 3.896), p = 0.0013), that supply complex I with NADH, but not alpha-ketoglutarate and malate (FIG. 2C- 2E). dOCR was not decreased following succinate injection, which supplies complex II with FADH (FIG. 2F). Furthermore, prior corticosterone exposure reduced EEC complex I enzyme activity, as determined by oxidation of NADH to NAD+ by complex I (t(8) = 3.446, p = 0.0087) (FIG. 2G). Additionally, Ndufa1 protein, a subunit required for complex I association with the mitochondrial inner membrane and proton translocation was reduced in EECs with prior corticosterone exposure (t(5) = 2.837, p = 0.0364) (FIG.2H). Example 3 Significant increases in H3K27me3 binding distribution reveals that prior stress initiates reprogramming of chromatin modifications

[0055] In another exemplary method, as prior stress exposure reduced expression of a majority of DEGs at day 9 (FIG. 3A, 3B), one hypothesis was that there are lasting changes in H3K27me3 distribution, a ubiquitous transcriptional repressor previously identified as stress- responsive in the caput epididymis. Using the high efficiency epigenetic profiling approach, Cleavage Under Targets and Release Using Nuclease sequencing (CUT&RUN) (schematic represented in FIG.3C), more than 50% of H3K27me3-binding loci were found associated with gene promoter regions (FIGS. 3D and FIG. 8A) and within 5kB of the transcription start site (FIG.3E). Differential binding analysis using the Diffbind package revealed 7282 differentially bound regions by H3K27me3 at the day 9 timepoint of an in vitro model (FIG.1A), representing 7135 enriched regions and only 147 H3K27me3 depleted regions (FIGS. 3F, 3G, FIG. 8B). Furthermore, H3K27me3 bound loci were associated with genes involved in mitochondrial organization among other metabolic and catabolic processes as determined by Gene Set Enrichment Analysis (FIG.3H). Example 4 Stress-sensitive EEC EV cargo convey intercellular signals that increase sperm cellular energy

[0056] In another method, because EVs serve as key intercellular signaling particles, roles of stress-mediated changes in EEC allostatic set point were examined that alter bioactive EV cargo regulating mitochondria in target sperm cells. (As used herein, cargo can refer to all components delivered by the EV to a target cell, for example, protein, RNA, DNA, etc.) Nanoparticle tracking 18 95743809.1106549-802378-CU6205H-02-PCT1 analysis revealed that conditioned EVs isolated from the media of EECs with prior corticosterone exposure (stress-EVs, conditioned EVs) were characterized by smaller median EV size (t(14) = 2.945, p = 0.0106) (FIGS. 4A, 4D). No difference was observed in the area under the curve analysis of the EV size distribution (FIGS.4A-4C). Whole cell respirometry demonstrated that EEC exposure to stress-EVs (FIG. 9A), decreases day 9 basal mitochondrial respiration (W(2.000, 46.90) = 2.558, p=0.0232, with the Dunnett’s post-hoc test between vehicle- and stress- EVs at p = 0.0187), ATP production rate (F(2, 62) = 4.411, p = 0.0162, with the Tukey post-hoc test between vehicle- and stress-EVs at p = 0.0125), and oxidative ATP production rate (W(2.000, 40.01) = 4.770, p = 0.0139, with the Dunnett’s post-hoc test between vehicle- and stress-EVs at p = 0.0104) (FIGS.9B-9D).

[0057] Given the influence of stress-mediated changes in EV cargo on EEC respiration and the known role for EEC EVs in sperm maturation, effects of EV cargo on sperm mitochondrial respiration were observed by incubating cauda sperm with stress- (or conditioned-, as used herein) EVs and control EVs prior to whole cell respirometry for analysis (FIG.4E). Incubating sperm with stress-EVs increased basal mitochondrial respiration (F(2, 17) = 5.593, p = 0.0136, with the Tukey post-hoc test between vehicle- and stress-EV exposure at p = 0.0434 and control and stress- EVs at p = 0.0180) (FIG.4F), extracellular acidification rate (F(2, 17) = 65.34, p < 0.0001, with the Tukey post-hoc test between vehicle- and stress-EV exposure at p < 0.0001 and control and stress-EV exposure at p < 0.0001) (FIG.4G), ATP production rate (F(2, 17) = 10.68, p = 0.0010, with the Tukey post-hoc test between vehicle- and stress-EV exposure at p = 0.0056 and control and stress-EV exposure at p = 0.0015) (FIG.4H), and glycolytic ATP production rate (F(2, 17) = 72.35, p < 0.0001, with the Tukey post-hoc test between vehicle- and stress-EV exposure, and control and stress-EV exposure at p < 0.0001) (FIG.4I). Sperm velocity is increased following conditioned-EV mediated increases in respiration

[0058] To assess the functional impact of conditioned-EV mediated increases in mitochondrial respiration, Computer Assisted Sperm Analysis (CASA) was used to evaluate mouse sperm motility parameters after conditioned-EV incubation (FIG. 5A). CASA revealed conditioned-EV exposure increased sperm curve velocity (t(13) = 3.532, p = 0.0037), average path velocity (t(13) = 2.752, p = 0.0165), and linear velocity (t(13) = 3.023, p = 0.0098) (FIG 5B- 5D). No differences were observed in the percent of motile sperm (t(13) = 1.444, p = 0.1724), 19 95743809.1106549-802378-CU6205H-02-PCT1 either progressive (t(13) = 0.3698, p = 0.7175) or non-progressive (t(13) = 1.475, p = 0.1639), or percent of immotile sperm (t(13) = 1.444, p = 0.1724) (FIG.5E-5H). Materials and Methods

[0059] Animals. Male C57BL / 6J and female 129S1 / SvImJ mice were obtained from Jackson Laboratories and were used to produce C57BL / 6:129 hybrids. All mice were housed in a 12:12 light:dark cycle with temperature 22°C and relative humidity 37%. Food (Teklad 2920X irradiated rodent diet, 19.4% protein, 47.0% carbohydrate, 6.5% fat) and water were provided ad libitum. All studies were performed according to experimental protocols approved by the University of Colorado Institutional Animal Care and Use Committee, and all procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

[0060] Tissue collection: Sires were rapidly decapitated under isoflurane anesthesia. To obtain sperm, M2 media (Sigma, M7167) was supplemented with 1 mM ZnCl (Sigma, Z0152) and equilibrated to 5% CO2 at 37°C (pH=6.7) in a droplet contained in mineral oil (Sigma, M5904). Caudal epididymis was removed, minced in the supplemented M2 media droplet, and incubated at 37°C, 5% CO2 for 45 minutes. Sperm were collected from the droplet with wide bore pipette tips. Sperm counted using a hemocytometer, centrifuged for 2 minutes at 400xg, and the pellet was resuspended in M2 media supplemented with 2 x 109EVs per million sperm. Sperm were incubated with EV supplemented or control media for 0.5h, pelleted by centrifugation at 400xg for 3 minutes and resuspended in Seahorse XF Base Media (Agilent, 103334-100) supplemented with 5.6 mM glucose (Gibco, A2494001), 1 mM pyruvate (Gibco, 11360070), 2 mM glutamine (Gibco, 25030081), 3 mg / mL fatty acid free bovine serum albumin (FisherScientific, BP9704-100) at a concentration of 1 million sperm per 50 uL media.

[0061] Cell culture and corticosterone treatment: Immortalized mouse distal caput epididymal epithelial (DC2) cells were purchased from Applied Biological Materials and cultured as previously described. Briefly, cells were seeded in on plates coated collagen type 1, rat tail (Millipore, C3867). Cells were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, 12440061) supplemented with 10% fetal bovine serum (Gibco, 26140079) and 1% penicillin- streptomycin (Gibco, 15070063). At monolayer confluency, the media was replaced, and cells were either treated with 1:1000 vehicle (ethanol; resulting in 0.1% ethanol) or 1:1000 corticosterone in ethanol (Cayman Chemical, 16063; 1.4 mM, resulting in 500 ng / ml of corticosterone). The media was replaced 72 hours (day 3) and 144 h (day 6) following the 20 95743809.1106549-802378-CU6205H-02-PCT1 treatment. Depending on the experiment, cells were collected at media changes prior to treatment, immediately following treatment on day 3, after 3 days of recovery on day 6, or after 6 days of recovery on day 9. For cell collection, cells were trypsinized in 0.25% trypsin-EDTA (Gibco), centrifuged at 500xg for 3 min, and frozen at -80°C until further analysis.

[0062] RNA isolation: Total RNA extraction from DC2 cell pellets was done using the TRIzol reagent (Thermo Fisher, 15596026) according to manufacturer’s protocol.

[0063] mRNA sequencing: Total RNA from DC2 cell pellets were quantified on a NanoDrop 2000 spectrophotometer (Thermo Scientific). Libraries for RNA-seq were made using a TruSeq Stranded mRNA Sample Preparation Kit (Illumina) with 500 ng RNA. All library sizes and concentrations were confirmed on a TapeStation 4200 (Agilent) and Qubit 3.0 Fluorometer (Thermo Fisher). Individually barcoded libraries were pooled and sequenced on an Illumina NextSeq 500 (75-bp single-end). FASTQ files were processed for pseudoalignment and abundance quantification of reads to the Mus musculus reference transcriptome (version 38) using Kallisto.

[0064] Lentiviral particle transduction and shRNA knockdown confirmation by RT-qPCR: Approximately 9,000 DC2 cells were seeded per well in a 96 well plate. At 80% confluency, DC2 cells were exposed to 18,000 transducing units of viral particles in culture media containing 8 ug / mL hexadimethrine bromide. Cultures were incubated with particles overnight. MISSION TRC2 pLKO.5-puro Empty Vector Control Transduction Particles (Sigma, SHC201V) (Empty Vector) was used to control for the effects of transduction, MISSION TRC2 pLKO.5-puro Non- Mammalian shRNA Control Transduction Particles (Sigma, SHC216V) were used to control for the expression of shRNA, and MISSION shRNA Lentiviral Clone Oligo TRCN0000026223 (Sigma, SHCLNV) was used to express shRNA to knockdown GR. Media was exchanged after the overnight incubation with complete culture medium. After 24 hours, the media was changed to selection media containing 2 ug / mL puromycin dihydrochloride (Sigma-Aldrich, P9620). Selection media was exchanged after three days, and cells were passaged into six well plates after six days of puromycin exposure when non-transduced cells growing in selection media were no longer viable. Selection media was exchanged every three days and cells were passaged as necessary. After 12 days, cells were passaged to 10 cm plates and seeded at 500 cells / plate. Three days later, colonies were selected with the agarose method (Lindberg Lab online protocol, http: / / thelindberglab.com / cloning-cells-with-the-agarose-method / ) and seeded in 48 well plates. Selection media was exchanged within 24 hours with conditioned selection media containing 20% 21 95743809.1106549-802378-CU6205H-02-PCT1 FBS. Then, selection media (10% FBS) was exchanged every three days. Colonies were passaged to six well plates after 9-12 days. Cells were maintained and passaged as necessary in selection media until they were cryopreserved in DC2 freeze media containing 20% FBS, 10% DMSO (Cell Signaling Technology, 12611), and 70% IMDM 19 days following agarose selection. For experiments, cells were thawed, cultured to confluency in selection media and seeded into experimental plates to be maintained in selection media throughout experiments.

[0065] To confirm glucocorticoid receptor (Nr3c1) mRNA knockdown, RNA was isolated using TRIzol reagent (Thermo Fisher, 15596026) according to the manufacturer’s protocol. RNA was eluted in RNAase free water. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814) according to the manufacturer’s protocol. Expression was quantified using reverse-transcription quantitative real-time PCR (RT-qPCR) in conjunction with TaqMan Assays (Applied Biosystems, 4444556) according to the manufacturer’s protocol. Briefly, each reaction was run in triplicate and each sample was probed for Nr3c1 (ThermoFisher Scientific, Assay ID: Mm00433832_m1, Catalog #4331182) and the endogenous 18S rRNA control (Applied Biosystems, 4332641). Reactions were run on a QuantStudio 5 Real- Time PCR System, and Ct values were calculated using the instrument’s onboard software. The mean Ct values for the endogenous control was subtracted from the corresponding Ct values of Nr3c1. The resulting ∆Ct values were used to calculate expression relative to the non-targeted shRNA control using the ∆∆Ct method.

[0066] Extracellular vesicle isolation: EVs were harvested from cultures seeded in 175 cm2flasks in medium supplemented with EV-depleted FBS (System Biosciences, EXO-FBS-50A-1). EVs were isolated by differential ultracentrifugation from conditioned media collected 72 hours after the last media change, six days after the corticosterone exposure ended. Cellular debris was removed from the media by centrifugation at 2,000 x g for 10 minutes at 4°C in a A-4-44 swing- bucket rotor (Eppendorf) followed by 9,000 rpm (avg 10,000 x g) for 35 minutes at 4°C using the Beckman Coulter LE-80 Ultracentrifuge and SW 32 Ti Swinging-Bucket Rotor (Beckman Coulter), which was used for all remaining ultracentrifugation. EVs were pelleted by ultracentrifugation at 28,500 rpm (average 100,000 x g) for 100 minutes at 4°C. Supernatant was removed by aspiration and the pellet was resuspended in 30 mL cold, filtered (0.22 µm) PBS. Ultracentrifugation at 28,500 rpm (avg 100,000 x g) for 100 minutes at 4°C was repeated and the supernatant was aspirated, leaving less than 1 mL PBS that the pellet was resuspended in. The 22 95743809.1106549-802378-CU6205H-02-PCT1 resulting volume of EVs was measured and recorded before freezing at -80°C. Samples were thawed on ice, diluted 1:500 in cold filtered (0.22 µm) water and analyzed using a ZetaView (Particle Metrix). Following analysis, samples were frozen at -80°C until use. Aliquots of the non- conditioned media were processed to serve as controls for potential EV contamination from culture media.

[0067] Nanoparticle tracking analysis (NTA): The concentration and size of EV aliquots were measured using a ZetaView BASIC Nanoparticle Tracking Analysis Microscope (Particle Metrix). EVs were diluted in freshly filtered (0.22 µm) water to achieve ~200 particles per frame. Size and concentration of particles were measured by scanning 11 cell positions with 30 frames per position, over 2 cycles. Surface charge was measured across 11 positions. Video acquisition sensitivity was set to 80 and shutter speed was set to 100. Videos were analyzed with the Native ZetaView software (version 8.05.14). Minimum brightness was set to 20, minimum area to 10, and maximum area to 1000. Comparisons of particle concentration (AUC), size, and zeta potential were analyzed by unpaired t tests. Statistical analyses and visualizations were performed using Graphpad Prism (version 9.5.0).

[0068] Respirometry: Respiration reported as oxygen consumption rates (OCR) were measured in intact adherent DC2 cells with a Seahorse Extracellular Flux (XFe96) Analyzer (Agilent). Cells were seeded at 3 million cells per well in Seahorse XF96 V3 PS Cell Culture Microplates (Agilent, 101085-004) and treated with corticosterone as described before performing cell-based respirometry performed after six days of recovery from treatment. Cell culture medium was replaced with Seahorse XF Base Medium (Agilent) supplemented with 15 mM glucose (Gibco, A2494001), 1 mM sodium pyruvate (Gibco, 11360070), and 4 mM L-glutamine (Gibco, 25030081), pH 7.4, unless indicated otherwise and incubated in a non-CO2 incubator at 37°C for no longer than one hour. Cells underwent a second wash with the respiration within one hour before taking measurements. The cartridge provided in the XFe96 FluxPak (Agilent, 102416-100) was hydrated in water overnight before calibrating with Seahorse XF Calibrant Solution (Agilent, 103681-100). For the Mito Stress Test (Agilent, 103015-100), the cartridge was loaded with 10x reagents in respiration media that would provide final concentrations of 1.5 uM oligomycin, 2 uM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and 0.5 uM Rotenone / Antimycin A (Agilent) after injection. For experiments in intact cells with additional injections, 10X solutions of sodium pyruvate (Gibco, 11360070) and L-glutamine (Gibco, 25030081) were prepared in 23 95743809.1106549-802378-CU6205H-02-PCT1 respiration media before loading into the cartridge injection ports. In all cases of adherent DC2 cells, the injection protocol required 3 min mix, 0 min wait, and 3 min measure for 3 cycles per baseline or injection.

[0069] To incubate DC2 cells with EVs isolated from DC2 media, DC2 cells were seeded in Seahorse XF96 microplates and treated with corticosterone as described above, with the exception that DC2 growth medium was supplemented with EV-depleted FBS (Systems Biosciences, EXO- FBS-50A-1). Three days after the corticosterone exposure ended, culture medium was supplemented with 3.14x108DC2 EVs / mL in PBS, or an equal volume of PBS control for three days. At the end of the EV exposure, culture medium was replaced with supplemented Seahorse XF Base Medium and respirometry was assayed as described above.

[0070] In another exemplary method, for experiments in need of permeabilization of adherent DC2 cells during analyzer measurements, cells were washed, substrates were prepared, and respiration was measured in a low potassium, low calcium buffer (3.5 mM KCl, 120 mM NaCl, 0.4 mM KH2PO4, 1.2 mM Na2SO4, 2 mM MgCl2, 20 mM Na-N-Tris-(hydroxymethyl)-methyl-2-amino- ethanesulfonic acid (TES pH 7.4), 1 mM EGTA, 0.4% wt / volume fat-free bovine serum albumin (BSA)). 10X solutions of the recombinant perfringolysin O (rPFO) and substrate combinations (malic acid and alpha-ketoglutarate, malic acid and sodium pyruvate, malic acid and L-glutamic acid, and succinic acid alone) were prepared in the low potassium, low calcium buffer with final diluted concentrations of 5 nM rPFO, and 10 mM alpha-ketoglutarate, malic acid, sodium pyruvate, L-glutamic acid, and succinic acid. For all cases of permeabilization, the injection protocol required 0.5 min mix, 1 minute wait, 3 min measure for 3 cycles per baseline or injection.

[0071] Following respirometry wells from each group which had no injections were washed twice with cold phosphate buffered saline (PBS) before lysing cells with RIPA Lysis Buffer prepared in cold PBS with an EDTA-free protease inhibitor (Roche) for 5 minutes. Protein concentration for normalization was quantified from these wells using the Pierce BCA Protein Assay Kit (Thermo Scientific) and analyzed on an Infinite M1000 plate reader (Tecan) using Magellan software (Tecan). Protein was averaged across the wells in each group for normalization.

[0072] To measure mitochondrial respiration in sperm, Seahorse XF96 microplates were coated with 0.5 mg / mL concanavalin A (Sigma) in PBS for 1 hour before removing the coating and allowing the plate to dry overnight at room temperature as previously reported. Reagents for the Mito Stress Test were prepared and loaded into the cartridge in the same media as the sperm, at 24 95743809.1106549-802378-CU6205H-02-PCT1 the concentrations described above with the exception that the appropriate final concentration of FCCP was determined to be 1 uM. Following a half an hour EV incubation (2 x 109EVs per million sperm in M2 media as described above), sperm were resuspended 1 million sperm per 50 uL media in warm Seahorse XF Base Media supplemented with 5.6 mM glucose, 1 mM pyruvate, 2 mL glutamine, 3 mg / mL fat free BSA, pH 6.9. Sperm were plated 1 million sperm / well and centrifuged at 250xg for 1 minute to collect sperm at the bottom of the well before carefully adding media to 200 uL per well. Sperm were visualized using a light microscope (Nikon) to ensure even distribution at the bottom of the well prior to plate loading. Equilibration of the Seahorse XFe Extracellular Flux Analyzer (Agilent) was completed while sperm were plated to reduce the time sperm were exposed to assay media prior to whole cell respirometry. Oxygen consumption rate and extracellular acidification rate of sperm were measured with 1-minute mix and 2-minute measure cycles at baseline and following injection of each component of the Mito Stress Test Kit. Three cycles, and therefore three measurements, were recorded for each step. Total cell count per well was used for normalization.

[0073] Respiration calculations following the Mito Stress Test were performed following previously published recommendations by the manufacturer and others. In assays requiring permeabilization with (rPFO) the difference in oxygen consumption rate (dOCR) between peak respiration following addition of substrates necessary for mitochondrial complex I or II function and the minimum respiration following permeabilization was calculated.

[0074] Complex I Enzyme Activity: Activity was measured in cells seeded in 96 well black walled, transparent bottom plates following the procedures outlined in the Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721) on an Infinite M1000 plate reader (Tecan) and analyzed using Magellan software (Tecan). Protein concentration for normalization was quantified by Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Cells were assayed six days after the described corticosterone exposure was removed.

[0075] Mitochondria Isolation and Protein Extraction: Mitochondria were isolated from fresh DC2 EECs following our treatment paradigm using a previously described protocol. Half of the sample was used for this protocol and half were pelleted and snap-frozen for nuclear extraction described below. Briefly, for mitochondria isolation, cell pellets were resuspended in an equal volume ice-cold hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM HEPES, pH 7.5) and allowed to swell for 10 minutes. Cells were homogenized on ice using 20 strokes of the dounce 25 95743809.1106549-802378-CU6205H-02-PCT1 homogenizer B pestle to produce greater than 50% lysed cells.5X extraction buffer (50 mM HEPES pH 7.5, 1M mannitol, 350 mM sucrose, 5 mM EGTA) was diluted to 2.5X and added to the sample to at 1X, one stroke was performed with the dounce homogenizer. The homogenizer was rinsed with 1X extraction buffer (1:8 buffer vol:sample vol), and the rinse was added to the sample. The homogenate was then centrifuged at 4°C at 1300xg for 5 minutes to remove nuclei and cell debris. This centrifugation was repeated twice with the resulting supernatant. The final supernatant was centrifuged at 4°C at 17000xg for 15 minutes resulting in the mitochondrial pellet. The supernatant was discarded, and the mitochondrial pellet was resuspended in 1 mL 1X extraction buffer before centrifuging repeating the 15-minute centrifugation at 17,000xg, 4°C. The resulting supernatant was aspirated, and the final mitochondrial pellet was snap-frozen in liquid nitrogen and stored at -80°C. Samples were thawed on ice for protein extraction with ice- cold RIPA lysis buffer (EMD Millipore Corp.) prepared with 1X cOmplete EDTA-free protease inhibitor (Roche). Mitochondrial pellets were resuspended in 150 µL cold lysis buffer, vortexed, incubated on ice for 5 minutes, and centrifuged at 8000xg for 10 minutes at 4°C to pellet any debris. The supernatant was collected, and protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) before storing at -80°C. Protein samples were stored until use in western immunoblot.

[0076] Nuclear Isolation and Protein Extraction: Isolation of nuclei were performed using the Nuclei EZ Prep Kit (Sigma, NUC101). Briefly, DC2 EEC pellets were thawed on ice, resuspended in 2 mL cold Nuclei EZ lysis buffer and incubated for 5 minutes on ice per the manufacturer instructions.1.5 mL of the sample was homogenized using 5 strokes of the dounce homogenizer pestle B on ice. The homogenizer was rinsed with 500 uL of the Nuclei EZ lysis buffer which was added to the sample before centrifuging for 5 minutes at 500xg at 4°C. The supernatant was removed, and the pellet was washed with 1 mL Nuclei EZ lysis buffer before centrifugation at 500xg for 5 minutes at 4°C. The supernatant was removed, and the pellet was washed with 1 mL cold PBS followed by another 5-minute centrifugation step. For protein extraction, the final pellet was resuspended in 200 uL RIPA lysis buffer with protease inhibitor, prepared as described above, shaken at 3000 rpm on the Disrupter Genie (Scientific Industries) for 15 minutes at 4°C, and centrifuged at 1600xg for 10 minutes at 4°C. The supernatant was collected, and protein was quantified using the Pierce BCA Protein Assay Kit (Thermo 26 95743809.1106549-802378-CU6205H-02-PCT1 Scientific, 23227) before storing at -80°C. Protein samples were stored until use in western immunoblot.

[0077] Whole Cell Lysate Protein Extraction: DC2 EEC cell pellets were thawed on ice and resuspended in 2 mL RIPA buffer with protease inhibitor prepared as described above. The mixture was rotated end over end for 15 minutes at 4°C, and centrifuged at 14000xg for 15 minutes at 4°C. The supernatant was collected, and protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) before storing samples at -80°C until use in western immunoblot.

[0078] Western Immunoblotting: For mitochondrial and nuclear protein, 20 µg of either mitochondrial or nuclear protein per lane was denatured by heating at 95°C for 5 minutes before loading onto a Nu-PAGE 4-12% Bis-Tris pre-cast gel (Invitrogen) for gel electrophoresis. NuPAGE antioxidant (Invitrogen) was added to the Nu-PAGE Running Buffer (Invitrogen) prior to electrophoresis at 180V for 50 minutes at room temperature. Prior to preparing the transfer cassette, the PVDF membrane (Millipore) was activated in 100% methanol for 30 seconds and rinsed in prepared Nu-PAGE transfer buffer (Invitrogen). Protein was transferred at 35V for 60 minutes at 4°C. The membrane was rinsed with milliQ water and blocked with Intercept Blocking Buffer (Licor) for 1 hour at room temperature. Depending on the experiment, the membrane was incubated overnight at 4°C in Intercept Blocking Buffer with 0.1% Tween-20 and primary antibodies that included either anti-H3 antibody at 1 µg / mL concentration (Abcam, ab176842), anti-GR antibody at 1:500 dilution (Santa Cruz), or anti-ATP-5a antibody at 1:2000 dilution (Abcam). When antibodies of the same species were utilized on the same blot, the membrane was cut and incubated individually with each antibody. The membrane was rinsed with PBS with 0.1% Tween-20 for 5 minutes at room temperature, four times and then incubated with the appropriate secondary antibodies including either IRDye 800CW Donkey anti-Mouse IgG secondary antibody at 1:5000 dilution (Licor, 926-32212) or IRDye 680RD Donkey anti- Rabbit IgG secondary antibody at 1:5000 dilution (Licor, 926-68073).

[0079] For protein extracted from whole cell lysate and probed for Ndufa1, the same procedures were followed with the following exceptions.16 µg protein was loaded per well on a NuPAGE 12% Tris-Bis pre-cast gel. The gel was cut prior to transfer to ensure optimal transfer of proteins of interest of differing sizes. Protein was transferred to a nitrocellulose membrane (Invitrogen, LC2000) with the upper portion transferring for 40 minutes at 30V followed by 20 minutes at 27 95743809.1106549-802378-CU6205H-02-PCT1 35V, and while the bottom portion transferred for 40 minutes at 30V, both at 4°C. Each membrane was probed with either anti-alpha tubulin primary antibody at 1:1000 dilution (Sigma, T5168) or anti-Ndufa1 at 1:1000 (MWFE) primary antibody (generously donated by Dr. Nagendra Yadava) for one hour at room temperature. Following washes, membranes were incubated for one hour at room temperature with either IRDye 680RD Donkey anti-Mouse IgG secondary antibody at 1:2000 dilution (Licor, 926-68072) or IRDye 800CW Donkey anti-Rabbit IgG secondary antibody at 1:3000 dilution (Licor, 926-32213).

[0080] Cleavage Under Targets and Release Using Nuclease (e.g., CUT&RUN): The CUT&RUN protocol was adopted as previously described. Adherent DC2 cells were cultured and treated in 6-well plates and collected by trypsinization (Gibco). About 500,000 cells were collected for each antibody incubation (anti-H3K27me3 antibody, Sigma, 07-499; anti-Rabbit IgG, Antibodies Online). Cells were bound to concanavalin-A coated beads (Bangs Labs) and incubated with antibody overnight. On the second day, pA / G MNase fusion protein (Cell Signaling) was bound during an hour-long incubation at 4°C and activated with 2 mM CaCl2during a 30 min incubation at 0° C. Samples were incubated for 30 min at 37° C to release chromatin fragments into the supernatant. Supernatant was collected and DNA was extracted using phenol:chloroform:isoamyl alcohol. DNA concentration was determined using the High Sensitivity dsDNA Qubit Assay (Invitrogen) and fragment sizes were determined using the High Sensitivity D1000 ScreenTape Assay (Agilent). DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) with the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set I) (NEB). All library sizes and concentrations were confirmed on a TapeStation 4200 (Agilent) and Qubit 3.0 Fluorometer (Thermo Fisher). Individually barcoded libraries were pooled and sequenced on an Illumina NextSeq 500 (75-bp paired-end).

[0081] Transmission Electron Imaging (TEM): DC2 cultures were washed with PBS and fixed overnight in a solution of 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M PIPES buffer (pH 7.2) for an hour at room temperature and then left overnight at 4°C. After fixation, cells were washed in PIPES buffer, quenched in 50 mM glycine in 0.1M PIPES buffer for 15 minutes, scraped off the plate and collected by centrifugation. Cell pellets were enrobed in 2.5% low melting pint agarose, trimmed into 1 mm3blocks and post-fixed with 1% osmium tetroxide, 0.25% potassium ferrocyanide in 0.1M PIPES buffer for 1 hour on ice. After washing agarose 28 95743809.1106549-802378-CU6205H-02-PCT1 blocks were loaded in the mPrepS capsule, en block stained with 1% Uranyl acetate, dehydrated and infiltrated in Araldite resin in an automated specimen processor ASP-1000 (Microscopy Innovations). Samples were embedded in pure Araldite resin and polymerized at 60°C for 24-48 hours. Ultrathin sections at ~70 nm thickness were cut on a Leica UC6 ultramicrotome (Leica Microsystems), and examined in a FEI Tecnai T12 electron microscope operated at 80 kV. Digital images were acquired by using an AMT bottom mount CCD camera and AMT600 software. This work utilized EM sample preparation instruments that were purchased with funding from an NIH SIG grant (1S10RR26870-1) and a Department of Defense DURIP grant, Proposal No.70183-LSRIP. Mitochondria in micrographs taken at 6500x magnification were identified and analyzed in ImageJ using methods described previously.

[0082] Computer Assisted Sperm Analysis: Following about a half hour EV incubation (2 x 109EVs per million sperm in M2 media as described above), sperm were resuspended 1 million sperm per 50 uL media in warm Seahorse XF Base Media supplemented with 5.6 mM glucose, 1 mM pyruvate, 2 mL glutamine, 3 mg / mL fat free BSA, pH 6.9. Sperm were plated 1 million sperm / well and kept on a plate warmer at 37°C before loading 5 uL into a prewarmed 20 µm chamber slide (CellVision, CV 1020-4CH) and analyzing sperm motility using the Sperm Class Analyzer (Microptics SL). This system consists of a microscope (Eclipse E600, Nikon) with a 10x objective under negative phase contrast (Nikon), a camera (Basler, acA1300-200uc), and a PC with the analysis software (SCA Version 6.6.15). Six randomly selected microscopic fields were captured (50 frames / second; 25 images per field) and analyzed by the software identifying immotile (<50 µm / s), slow-medium (>50 µm / s), and rapid (>200 µm / s), with progressive sperm defined by STR>50. Curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) were measured.

[0083] Statistics: Statistical analyses were performed using GraphPad Prism (version 9.5.0) with the exception of next-generation sequencing analyses. Respirometry, complex I enzyme function, western blot, EV AUC and size, CASA, and TEM data were analyzed by two-way ANOVAs or Student’s t-tests as appropriate using GraphPad Prism (version 9.5.0). Proportions of mitochondrial ultrastructure were analyzed using the chi square test. Outliers were identified using the Grubbs’ Test. When appropriate, Bonferroni’s or Sidak’s multiple comparisons, or Tukey test was used to explore main effects. Significance was set at p<0.05. For the DC2 RNA sequencing experiment, a 0.5 minimum log2-fold-change (lfc) cutoff was established, and significance was 29 95743809.1106549-802378-CU6205H-02-PCT1 determined by false discovery rate (FDR) of <0.05. For the WGCNA, gene ontology for Biological Processes with FDR<0.01 were reported. For CUTNRUN sequencing analysis, differential binding was determined by FDR<0.05 in the CUTNRUN sequencing experiment.

[0084] Bioinformatics analyses: Analyses were performed using R version 4.2.2 and Bioconductor version 3.16.0. Additionally, data from CUTNRUN were visualized using the usegalaxy.org online platform.

[0085] DC2 differential expression analysis. Our DC2 RNA sequencing pipeline was designed using the DIY Transcriptomics framework. Briefly, Bioconductor package tximport (version 1.26.1) was used to collapse gene isoforms to gene symbols. Genes included in the analysis had >1 counts per million in at least 4 samples and gene expression distributions were normalized using Bioconductor package edgeR (version). All heatmaps are plotted as average Z scores per treatment group and arranged through hierarchical clustering of groups. The R package Stats (version 4.2.2) was used for principal component analysis and to cluster co-regulated genes (method= “spearman”, method= “complete”). Heatmaps were generated using R package pheatmap (version 1.0.12). The limma Bioconductor package (version 3.54.0) function voom was used for normalization. limma was also used to test normalized counts for differential abundance. The heatmap of differentially expressed genes between day 9 vehicle and corticosterone groups was generated using the heatmap.2 function of the R package gplots (version 3.1.3).

[0086] CUT&RUN Differential Binding Analysis (DBA): Briefly, alignment and data processing was performed following the protocols previously disclosed. Briefly, paired-end reads from the FASTQ files were aligned using Bowtie2 (version 2.4.2) to the Genome Reference Consortium Mouse Build 38 and the iGenomes Escherichia coli strain K12, MG1655 Build 2001-10-15 (Illumina) to allow for calibration based on carry over E.coli DNA introduced with the pA / G MNase using the py_sam_2_spikenormbg.py script. Peak calling was performed using the py_peak_calling.py script. Both the normalization and peak calling scripts are available. Differential binding analysis was performed using the Bioconductor package DiffBind (version 2.15.1). Peaks present in a minimum of 4 peaksets with score=DBA_SCORE_TMM_MINUS_FULL set in dba.count, using method=DBA_EDGER in dba.analyze. Peaks with differential binding determined by FDR < 0.05 were retained. Bioconductor packages ChIPpeakAnno and ChIPseeker were used to perform peak annotation, generate bar and distance to transcription site plot. Heatmaps of representative samples were 30 95743809.1106549-802378-CU6205H-02-PCT1 generated by uploading the data to the Galaxy web platform and using the public server at usegalaxy.org to analyze the data with the deeptools toolkit. Peaks were visualized using the UCSC Genome Browser. ****************************************** All the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations can be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims. 31 95743809.1

Claims

106549-802378-CU6205H-02-PCT1 WHAT IS CLAIMED IS:

1. A composition comprising, conditioned extracellular vesicles (EVs); at least one sperm, and a medium.

2. The composition according to claim 1, wherein the conditioned EVs are derived from epididymal epithelial cells (EECs) exposed for a predetermined period to at least one glucocorticoid.

3. The composition according claim 2, wherein the at least one glucocorticoid comprises at least one of cortisone, cortisol, corticosterone, other corticosteroid type agent, hydrocortisone, prednisone, dexamethasone or other known glucocorticoid capable of inducing stress or a stress environment.

4. The composition according to claims 2 or 3, wherein the conditioned EVs are post harvested EECs after exposure to at least one glucocorticoid for about one hour to about one week and then incubated for at least one hour to about 2 weeks without the at least one glucocorticoid.

5. The composition according to any one of claims 1-4, wherein the conditioned EVs have increased expression of R-spondin family proteins (RSPO family) compared to un-conditioned EVs.

6. The composition according to any one of claims 1-5, wherein at least one sperm of the at least one sperm in the conditioned EV composition have increased mitochondrial ATP production by about 1.1 to about 2.5-fold compared sperm in the presence of un-conditioned EVs.

7. The composition according to any one of claims 1-6, wherein the conditioned EVs have increased conditioned EV-sperm fusion compared to unconditioned EVs.

8. The composition according to any one of claims 1-7, wherein the sperm are human sperm. 32 95743809.1106549-802378-CU6205H-02-PCT1 9. The composition according to any one of claims 1-7, wherein the sperm are non-human animal sperm.

10. The composition according to any one of claims 1-7, wherein the sperm are food animal, companion animal, pet, or livestock sperm.

11. The composition according to any one of claim 1-7, wherein the sperm are bird sperm or aquatic species sperm.

12. The composition according to any one of claims 1-11, further comprising at least one additional agent comprising an agent to enhance sperm motility or preserve sperm viability.

13. An in vitro method for improving sperm motility comprising: obtaining conditioned EVs from EECs; and introducing the conditioned EVs to sperm in a composition comprising a medium.

14. The method according to claim 13, wherein the conditioned EVs comprise EVs exposed to at least one glucocorticoid for about 1 hour to about 1 week and then incubated for at least one hour to about 2 weeks without the at least one glucocorticoid.

15. The method according to claim 13 or 14, further comprising introducing at least one replacement media to the EVs after the EVs are exposed to the at least one glucocorticoid.

16. The method according to any one of claims 13-15, further comprising introducing at least one additional agent to the composition.

17. The method according to any one of claims 13-16, further comprising incubating the sperm with the conditioned EVs for about 1 minute to about 1 day.

18. The method according to any one of claims 13-17, wherein the sperm exposed to conditioned EVs for a predetermined period are used in an artificial insemination process. 33 95743809.1106549-802378-CU6205H-02-PCT1 19. An in vitro method for creating conditioned EVs comprising: obtaining a plurality of EVs from EECs; exposing the plurality of EVs to at least one glucocorticoid in a media for about one hour to about 1 week; and introducing at least one glucocorticoid free media to the EVs and further incubating the EVs in the glucocorticoid free media for about one hour to about 2 weeks and creating conditioned EVs.

20. The method according to claim 19, wherein the EVs without the at least one glucocorticoid are incubated for about 3 hours to about 1 day.

21. The method according to claim 19 or 20, further comprising harvesting the conditioned EVs.

22. A kit comprising, at least one composition according to claims 1-12; and at least one container.

23. An in vitro method for improving fertility in a subject comprising, providing sperm of a composition according to any one of claims 1-12 to an egg of a subject in an in vitro environment to improve fertilization of the egg.

24. The method according to claim 23, further comprising introducing the fertilized egg to a subject for maturation or incubating the fertilized egg in an artificial environment. 34 95743809.1

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