Long-term Hyperandrogenemia and/or Western-style Diet in Rhesus Macaque Females Impairs Preimplantation Embryogenesis.

Hyperandrogenemia and obesity are common in women with polycystic ovary syndrome, but it is currently unclear how each alone or in combination contribute to reproductive dysfunction and female infertility. To distinguish the individual and combined effects of hyperandrogenemia and an obesogenic diet on ovarian function, prepubertal female rhesus macaques received a standard control (C) diet, testosterone (T) implants, an obesogenic Western-style diet (WSD), or both (T + WSD). After 5 to 6 years of treatment, the females underwent metabolic assessments and controlled ovarian stimulations. Follicular fluid (FF) was collected for steroid and cytokine analysis and the oocytes fertilized in vitro. Although the T + WSD females exhibited higher insulin resistance compared to the controls, there were no significant differences in metabolic parameters between treatments. Significantly higher concentrations of CXCL-10 were detected in the FF from the T group, but no significant differences in intrafollicular steroid levels were observed. Immunostaining of cleavage-stage embryos revealed multiple nuclear abnormalities in the T, WSD, and T + WSD groups. Single-cell DNA sequencing showed that while C embryos contained primarily euploid blastomeres, most cells in the other treatment groups were aneuploid. Despite yielding a higher number of mature oocytes, T + WSD treatment resulted in significantly reduced blastocyst formation rates compared to the T group. RNA sequencing analysis of individual blastocysts showed differential expression of genes involved in critical implantation processes between the C group and other treatments. Collectively, we show that long-term WSD consumption reduces the capacity of fertilized oocytes to develop into blastocysts and that the addition of T further impacts gene expression and embryogenesis.

Metabolomics analysis of follicular fluid coupled with oocyte aspiration reveals importance of glucocorticoids in primate periovulatory follicle competency.

Gonadotropin administration during infertility treatment stimulates the growth and development of multiple ovarian follicles, yielding heterogeneous oocytes with variable capacity for fertilization, cleavage, and blastocyst formation. To determine how the intrafollicular environment affects oocyte competency, 74 individual rhesus macaque follicles were aspirated and the corresponding oocytes classified as failed to cleave, cleaved but arrested prior to blastulation, or those that formed blastocysts following in vitro fertilization. Metabolomics analysis of the follicular fluid (FF) identified 60 unique metabolites that were significantly different between embryo classifications, of which a notable increase in the intrafollicular ratio of cortisol to cortisone was observed in the blastocyst group. Immunolocalization of the glucocorticoid receptor (GR, NR3C1) revealed translocation from the cytoplasm to nucleus with oocyte maturation in vitro and, correlation to intrafollicular expression of the 11-hydroxy steroid dehydrogenases that interconvert these glucocorticoids was detected upon an ovulatory stimulus in vivo. While NR3C1 knockdown in oocytes had no effect on their maturation or fertilization, expansion of the associated cumulus granulosa cells was inhibited. Our findings indicate an important role for NR3C1 in the regulation of follicular processes via paracrine signaling. Further studies are required to define the means through which the FF cortisol:cortisone ratio determines oocyte competency.

Short-term Western-style diet negatively impacts reproductive outcomes in primates.

A maternal Western-style diet (WSD) is associated with poor reproductive outcomes, but whether this is from the diet itself or underlying metabolic dysfunction is unknown. Here, we performed a longitudinal study using regularly cycling female rhesus macaques (n = 10) that underwent 2 consecutive in vitro fertilization (IVF) cycles, one while consuming a low-fat diet and another 6-8 months after consuming a high-fat WSD. Metabolic data were collected from the females prior to each IVF cycle. Follicular fluid (FF) and oocytes were assessed for cytokine/steroid levels and IVF potential, respectively. Although transition to a WSD led to weight gain and increased body fat, no difference in insulin levels was observed. A significant decrease in IL-1RA concentration and the ratio of cortisol/cortisone was detected in FF after WSD intake. Despite an increased probability of isolating mature oocytes, a 44% reduction in blastocyst number was observed with WSD consumption, and time-lapse imaging revealed delayed mitotic timing and multipolar divisions. RNA sequencing of blastocysts demonstrated dysregulation of genes involved in RNA binding, protein channel activity, mitochondrial function and pluripotency versus cell differentiation after WSD consumption. Thus, short-term WSD consumption promotes a proinflammatory intrafollicular microenvironment that is associated with impaired preimplantation development in the absence of large-scale metabolic changes.

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination.

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.

Treatment of female rhesus macaques with a somatostatin receptor antagonist that increases oocyte fertilization rates without affecting post-fertilization development outcomes.

PURPOSE: To determine the effects of PGL1001, a somatostatin receptor isoform-2 (SSTR-2) antagonist, on ovarian follicle development, oocyte fertilization, and subsequent embryo developmental potential in the rhesus macaque. METHODS: Cycling female rhesus macaques (N = 8) received vehicle through one menstrual (control) cycle, followed by daily injections of PGL1001, a SSTR-2 antagonist, for three menstrual (treatment) cycles. Main endpoints include overall animal health and ovarian hormones (e.g., estradiol [E2], progesterone [P4], and anti-Müllerian hormone [AMH]), ovarian circumference, numbers of oocytes and their maturation status following controlled ovarian stimulation (COS), as well as oocyte fertilization and subsequent blastocyst rates that were assessed in control and PGL1001 treatment cycles. Circulating PGL1001 levels were assessed at baseline as well as 6, 60, and 90 days during treatment. RESULTS: PGL1001 treatment did not impact overall animal health, menstrual cycle length, or circulating levels of ovarian hormones (E2, P4, and AMH) in comparison to vehicle treatment during natural cycles. PGL1001 treatment increased (p Ë‚ 0.05) ovarian circumference and the day 8 to day 1 ratio of AMH levels (p Ë‚ 0.05) during a COS protocol, as well as oocyte fertilization rates compared to the vehicle treatment interval. Blastocyst development rates were not significantly different between vehicle and PGL1001 treatment groups. CONCLUSION: Prolonged treatment with PGL1001 appears to be safe and does not affect rhesus macaque general health, menstrual cycle length, or ovarian hormone production. Interestingly, PGL1001 treatment increased the fertilization rate of rhesus macaque oocytes collected following ovarian stimulation.

Leukemia Inhibitory Factor Is Necessary for Ovulation in Female Rhesus Macaques.

Although the requirement of pituitary-derived LH for ovulation is well documented, the intrafollicular paracrine and autocrine processes elicited by LH necessary for follicle rupture are not fully understood. Evaluating a published rhesus macaque periovulatory transcriptome database revealed that mRNA encoding leukemia inhibitory factor (LIF) and its downstream signaling effectors are up-regulated in the follicle after animals receive an ovulatory stimulus (human chorionic gonadotropin [hCG]). Follicular LIF mRNA and protein levels are below the limit of detection before the administration of hCG but increase significantly 12 hours thereafter. Downstream LIF receptor (LIFR) signaling components including IL-6 signal transducer, the receptor associated Janus kinase 1, and the transcription factor signal transducer and activator of transcription 3 also exhibit increased expression in the rhesus macaque follicle 12 hours after administration of an ovulatory hCG bolus. A laparoscopic ovarian evaluation 72 hours after the injection of a LIF antagonist (soluble LIFR) into the rhesus macaque preovulatory follicle and hCG administration revealed blocking LIF action prevented ovulation (typically occurs 36-44 h after hCG). Moreover, ovaries removed 52 hours after both hCG and intrafollicular soluble LIFR administration confirmed ovulation was blocked as evidenced by the presence of an intact follicle and a trapped cumulus-oocyte complex. These findings give new insight into the role of LIF in the primate ovary and could lead to the development of new approaches for the control of fertility.

Systematic analysis of protease gene expression in the rhesus macaque ovulatory follicle: metalloproteinase involvement in follicle rupture.

Protease genes were identified that exhibited increased mRNA levels before and immediately after rupture of the naturally selected, dominant follicle of rhesus macaques at specific intervals after an ovulatory stimulus. Quantitative real-time PCR validation revealed increased mRNA levels for matrix metalloproteinase (MMP1, MMP9, MMP10, and MMP19) and a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS1, ADAMTS4, ADAMTS9, and ADAMTS15) family members, the cysteine protease cathepsin L (CTSL), the serine protease urokinase-type plasminogen activator (PLAU), and the aspartic acid protease pepsinogen 5 (PGA5). With the exception of MMP9, ADAMTS1, and PGA5, mRNA levels for all other up-regulated proteases increased significantly (P < 0.05) 12 h after an ovulatory human chorionic gonadotropin (hCG) bolus. MMP1, -10, and -19; ADAMTS1, -4, and -9; CTSL; PLAU; and PGA5 also exhibited a secondary increase in mRNA levels in 36-h postovulatory follicles. To further determine metalloproteinase involvement in ovulation, vehicle (n = 4) or metalloproteinase inhibitor (GM6001, 0.5 µg/follicle, n = 8) was injected into the preovulatory follicle at the time of hCG administration. Of the eight GM6001-injected follicles, none displayed typical stigmata indicative of ovulation at 72 h after hCG; whereas all four vehicle-injected follicles ovulated. No significant differences in mean luteal progesterone levels or luteal phase length occurred between the two groups. Subsequent histological analysis revealed that vehicle-injected follicles ruptured, whereas GM6001-injected follicles did not, as evidenced by an intact stroma and trapped oocytes (n = 3). These findings demonstrate metalloproteinases are critical for follicle rupture in primates, and blocking their activity would serve as a novel, nonhormonal means to achieve contraception.

Dynamic changes in gene expression that occur during the period of spontaneous functional regression in the rhesus macaque corpus luteum.

Luteolysis of the corpus luteum (CL) during nonfertile cycles involves a cessation of progesterone (P4) synthesis (functional regression) and subsequent structural remodeling. The molecular processes responsible for initiation of luteal regression in the primate CL are poorly defined. Therefore, a genomic approach was used to systematically identify differentially expressed genes in the rhesus macaque CL during spontaneous luteolysis. CL were collected before [d 10-11 after LH surge, mid-late (ML) stage] or during (d 14-16, late stage) functional regression. Based on P4 levels, late-stage CL were subdivided into functional-late (serum P4 > 1.5 ng/ml) and functionally regressed late (FRL) (serum P4 < 0.5 ng/ml) groups (n = 4 CL per group). Total RNA was isolated, labeled, and hybridized to Affymetrix genome microarrays that contain elements representing the entire rhesus macaque transcriptome. With the ML stage serving as the baseline, there were 681 differentially expressed transcripts (>2-fold change; P < 0.05) that could be categorized into three primary patterns of expression: 1) increasing from ML through FRL; 2) decreasing from ML through FRL; and 3) increasing ML to functional late, followed by a decrease in FRL. Ontology analysis revealed potential mechanisms and pathways associated with functional and/or structural regression of the macaque CL. Quantitative real-time PCR was used to validate microarray expression patterns of 13 genes with the results being consistent between the two methodologies. Protein levels were found to parallel mRNA profiles in four of five differentially expressed genes analyzed by Western blot. Thus, this database will facilitate the identification of mechanisms involved in primate luteal regression.

Prostaglandin synthesis, metabolism, and signaling potential in the rhesus macaque corpus luteum throughout the luteal phase of the menstrual cycle.

Prostaglandins in the corpus luteum (CL) reportedly serve as luteotropic and luteolytic agents. Based mainly on studies conducted in domesticated animals and rodents, prostaglandin E2 (PGE2) is generally considered a luteotropic factor, whereas uterine-derived prostaglandin F2alpha (PGF2alpha) initiates luteolysis. However, the role of prostaglandins in regulating primate luteal structure-function is poorly understood. Therefore, a comprehensive analysis of individual mRNA or proteins that are involved in PGE2 and PGF2alpha biosynthesis, metabolism, and signaling was performed using CL obtained at distinct stages of the luteal life span during the menstrual cycle in rhesus monkeys. Peak levels of proteins involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 2, microsomal PGE2 synthase-1) and signaling (PGE2 receptor 3) occurred during periods corresponding to development and maintenance of the primate CL. Immunohistochemistry studies indicated that large luteal cells express PGE2 synthesizing and signaling proteins. Expression of PGE2 synthesizing and signaling proteins significantly decreased preceding the period of functional regression of the CL, which also coincided with increasing levels of PGF2alpha receptor protein expression within the large luteal cells. Moreover, significant levels of mRNA expression for several aldoketo reductase family members that synthesize PGF2alpha from other prostaglandins were observed throughout the rhesus macaque luteal phase, thus supporting the possibility of intraluteal PGF2alpha production. Collectively, our results indicate that there may be intraluteal synthesis and signaling of PGE2 during development and maintenance of the primate CL, followed by a shift to intraluteal PGF2alpha synthesis and signaling as the CL nears the time of luteolysis.

Systematic determination of differential gene expression in the primate corpus luteum during the luteal phase of the menstrual cycle.

The molecular and cellular processes required for development, function, and regression of the primate corpus luteum (CL) are poorly defined. We hypothesized that there are dynamic changes in gene expression occurring during the CL life span, which represent proteins and pathways critical to its regulation. Therefore, a genomic approach was utilized to systematically identify differentially expressed genes in the rhesus macaque CL during the luteal phase of natural menstrual cycles. CL were collected between d 3-5 (early stage), d 7-8 (mid), d 10-12 (mid-late), d 14-16 (late), or d 18-19 (very-late) after the midcycle LH surge. From the early through very-late stages, 3234 transcripts were differentially expressed, with 879 occurring from the early through late stages that encompass the processes of luteinization, maintenance, and functional regression. To characterize gene changes most relevant to these processes, ontology analysis was performed using the list of 879 differentially expressed transcripts. Four main groups of related genes were identified with relevance to luteal physiology including: 1) immune function; 2) hormone and growth factor signaling; 3) steroidogenesis; and 4) prostaglandin biosynthesis, metabolism, and signaling. A subset of genes representing each of the four major categories was selected for validation of microarray results by quantitative real-time PCR. Results in mRNA levels were similar between the two methodologies for 17 of 18 genes. Additionally, protein levels for three genes were determined by Western blot analysis to parallel mRNA levels. This database will facilitate the identification of many novel or previously underappreciated pathways that regulate the structure and function of the primate CL.

The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies.

Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75-99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.