Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism

A follicular spike in cyclic AMP (cAMP) and its subsequent degradation to AMP promotes oocyte maturation and ovulation. In vitro matured (IVM) oocytes do not receive the cAMP increase that occurs in vivo, and artificial elevation of cAMP in IVM cumulus-oocyte complexes improves oocyte developmental potential. This study examined whether mouse oocytes can use the cAMP degradation product AMP to generate ATP via the adenosine salvage pathway, and examined whether pharmacological elevation of cAMP in IVM cumulus-oocyte complexes alters ATP levels. Oocytes cultured with isotopic 13C5-AMP dose-dependently produced 13C5-ATP, however total cellular ATP remained constant. Pharmacological elevation of cAMP using forskolin and IBMX prior to IVM decreased oocyte ATP and ATP:ADP ratio, and promoted activity of the energy regulator AMPK. Conversely, cumulus cells exhibited higher ATP and no change in AMPK. Culture of oocytes without their cumulus cells or inhibition of their gap-junctional communication yielded lower oocyte 13C5-ATP, indicating that cumulus cells facilitate ATP production via the adenosine salvage pathway. In conclusion, this study demonstrates that mouse oocytes can generate ATP from AMP via the adenosine salvage pathway, and cAMP elevation alters adenine nucleotide metabolism and may provide AMP for energy production via the adenosine salvage pathway during the energetically demanding process of meiotic maturation.Dulama Richani, Cathy F. Lavea, Raji Kanakkaparambil, Angelique H. Riepsamen, Michael J. Bertoldo, Sonia Bustamante and Robert B. Gilchris

Pathogenesis of reproductive and metabolic PCOS traits in a mouse model

Polycystic ovary syndrome (PCOS) is a common and heterogeneous disorder; however, the etiology and pathogenesis of PCOS are poorly understood and current management is symptom-based. Defining the pathogenesis of PCOS traits is important for developing early PCOS detection markers and new treatment strategies. Hyperandrogenism is a defining characteristic of PCOS, and studies support a role for androgen-driven actions in the development of PCOS. Therefore, we aimed to determine the temporal pattern of development of PCOS features in a well-characterized dihydrotestosterone (DHT)-induced PCOS mouse model after 2, 4, and 8 weeks of DHT exposure. Following 2 weeks of treatment, DHT induced the key PCOS reproductive features of acyclicity, anovulation, and multifollicular ovaries as well as a decrease in large antral follicle health. DHT-treated mice displayed the metabolic PCOS characteristics of increased body weight and exhibited increased visceral adiposity after 8 weeks of DHT treatment. DHT treatment also led to an increase in circulating cholesterol after 2 weeks of exposure and had an overall effect on fasting glucose levels, but not triglycerides, aspartate transaminase (AST) and alanine transaminase (ALT) levels, or hepatic steatosis. These data reveal that in this experimental PCOS mouse model, acyclicity, anovulation, and increased body weight are early features of a developing PCOS phenotype whereas adiposity, impaired glucose tolerance, dyslipidemia, and hepatic steatosis are later developing features of PCOS. These findings provide insights into the likely sequence of PCOS trait development and support the addition of body weight criteria to the early diagnosis of PCOS.Valentina Rodriguez Paris, Melissa C. Edwards, Ali Aflatounian, Michael J. Bertoldo, William L. Ledger, David J. Handelsman, Robert B. Gilchrist, and Kirsty A. Walter

Multispectral autofluorescence characteristics of reproductive aging in old and young mouse oocytes

Published online: 24 February 2022Increasing age has a major detrimental impact on female fertility, which, with an ageing population, has major sociological implications. This impact is primarily mediated through deteriorating quality of the oocyte. Deteriorating oocyte quality with biological age is the greatest rate-limiting factor to female fertility. Here we have used label-free, non-invasive multi-spectral imaging to identify unique autofluorescence profiles of oocytes from young and aged animals. Discriminant analysis demonstrated that young oocytes have a distinct autofluorescent profile which accurately distinguishes them from aged oocytes. We recently showed that treatment with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) restored oocyte quality and fertility in aged animals, and when our analysis was applied to oocytes from aged animals treated with NMN, 85% of these oocytes were classified as having the autofluorescent signature of young animals. Spectral unmixing using the Robust Dependent Component Analysis (RoDECA) algorithm demonstrated that NMN treatment altered the metabolic profile of oocytes, increasing free NAD(P)H, protein bound NAD(P)H, redox ratio and the ratio of bound to free NAD(P)H. The frequency of oocytes with simultaneously high NAD(P)H and flavin content was also significantly increased in mice treated with NMN. Young and Aged + NMN oocytes had a smoother spectral distribution, with the distribution of NAD(P)H in young oocytes specifically differing from that of aged oocytes. Identifying the multispectral profile of oocyte autofluorescence during aging could have utility as a non-invasive and sensitive measure of oocyte quality.Jared M. Campbell, Saabah B. Mahbub, Michael J. Bertoldo, Abbas Habibalahi, Dale M. Goss, William L. Ledger, Robert B. Gilchrist, Lindsay E. Wu, Ewa M. Goldy

Unique Deep Radiomic Signature Shows NMN Treatment Reverses Morphology of Oocytes from Aged Mice

The purpose of this study is to develop a deep radiomic signature based on an artificial intelligence (AI) model. This radiomic signature identifies oocyte morphological changes corresponding to reproductive aging in bright field images captured by optical light microscopy. Oocytes were collected from three mice groups: young (4- to 5-week-old) C57BL/6J female mice, aged (12-monthold) mice, and aged mice treated with the NAD+ precursor nicotinamide mononucleotide (NMN), a treatment recently shown to rejuvenate aspects of fertility in aged mice. We applied deep learning, swarm intelligence, and discriminative analysis to images of mouse oocytes taken by bright field microscopy to identify a highly informative deep radiomic signature (DRS) of oocyte morphology. Predictive DRS accuracy was determined by evaluating sensitivity, specificity, and cross-validation, and was visualized using scatter plots of the data associated with three groups: Young, old and Old + NMN. DRS could successfully distinguish morphological changes in oocytes associated with maternal age with 92% accuracy (AUC~1), reflecting this decline in oocyte quality. We then employed the DRS to evaluate the impact of the treatment of reproductively aged mice with NMN. The DRS signature classified 60% of oocytes from NMN-treated aged mice as having a ‘young’ morphology. In conclusion, the DRS signature developed in this study was successfully able to detect aging-related oocyte morphological changes. The significance of our approach is that DRS applied to bright field oocyte images will allow us to distinguish and select oocytes originally affected by reproductive aging and whose quality has been successfully restored by the NMN therapy.Abbas Habibalahi, Jared M. Campbell, Michael J. Bertoldo, Saabah B. Mahbub, Dale M. Goss, William L. Ledger, Robert B. Gilchrist, Lindsay E. Wu, and Ewa M. Goldy

Estrus cycle monitoring of captive collared peccaries (Pecari tajacu) in semiarid conditions

Collared peccaries (Peccary tajacu) are among the most hunted species in Latin America due the appreciation of their pelt and meat. In order to optimize breeding management of captive born collared peccaries in semiarid conditions, the objective was to describe and correlate the changes in the ovarian ultrasonographic pattern, hormonal profile, vulvar appearance, and vaginal cytology during the estrus cycle in this species. During 45 days, females (n=4) were subjected each three days to blood collection destined to hormonal dosage by enzyme immunoassay (EIA). In the same occasions, evaluation of external genitalia, ovarian ultrasonography and vaginal cytology were conducted. Results are presented as means and standard deviations. According to hormonal dosage, six estrous cycles were identified as lasting 21.0 ± 5.7 days, being on average 6 days for the estrogenic phase and 15 days for the progesterone phase. Estrogen presented mean peak values of 55.6 ± 20.5 pg/mL. During the luteal phase, the high values for progesterone were 35.3 ± 4.4 ng/mL. The presence of vaginal mucus, a reddish vaginal mucosa and the separation of the vulvar lips were verified in all animals during the estrogenic peak. Through ultrasonography, ovarian follicles measuring 0.2±0.1 cm were visualized during the estrogen peak. Corpora lutea presented hyperechoic regions measuring 0.4±0.2 cm identified during luteal phase. No significant differences (P>0.05) between proportions of vaginal epithelial cells were identified when comparing estrogenic and progesterone phases. In conclusion, female collared peccaries, captive born in semiarid conditions, have an estral cycle that lasts 21.0±5.7 days, with estrous signs characterized by vulvar lips edema and hyperemic vaginal mucosa, coinciding with developed follicles and high estrogen levels

Seasonal effects on oocyte developmental competence in sows experiencing pregnancy loss

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Capacitation IVM improves cumulus function and oocyte quality in minimally stimulated mice

Published online: 31 October 2019PURPOSE:Oocyte in vitro maturation (IVM) is a patient-friendly reproductive technology but lower success rates than IVF have limited its uptake. Capacitation-IVM (CAPA-IVM) is an innovative new IVM system currently undergoing clinical evaluation. This study aimed to determine temporal effects of the pre-IVM phase of CAPA-IVM on cumulus function and oocyte developmental competence in mildly-stimulated mice. METHODS:Immature cumulus oocyte complexes (COCs) derived from mildly stimulated (23 h PMSG) 28-day-old mice underwent pre-IVM for 0-24 h in medium containing c-type natriuretic peptide (CNP), E2, FSH and insulin, prior to IVM (CAPA-IVM). The effect of pre-IVM duration on cumulus cell function and embryo development post-CAPA-IVM/IVF was assessed. RESULTS:Day 6 blastocyst rate increased incrementally with increasing pre-IVM duration: 40.6 ± 2.0%, 45.8 ± 1.2%, 52.2 ± 3.5%, 53.3 ± 5.9%, and 59.9 ± 2.5% for 0, 2, 6, 12, and 24 h pre-IVM, respectively (P < 0.01). DNA content/COC, a measure of cumulus cell proliferation, was significantly higher with 24 h pre-IVM group compared to 0, 2, or 6 h pre-IVM (P < 0.001). Pre-IVM for 24 h significantly increased cumulus expansion and mRNA expression of matrix genes Has2 and Tnfaip6 and Areg relative to no pre-IVM control (P < 0.01). Cumulus-oocyte gap-junctional communication (GJC) was maintained throughout 24 h pre-IVM (P < 0.0001), and GJC loss was slowed during the subsequent IVM phase, whilst meiotic resumption was accelerated (P < 0.05). Pre-IVM increased COC ATP and ADP content (P < 0.05), but not AMP, ATP/ADP, and energy charge. CONCLUSION:The pre-IVM phase of CAPA-IVM improves the quality of IVM oocytes in a temporally dependent manner and significantly influences cumulus cell function including increased cell proliferation, cumulus expansion, and prolonged cumulus-oocyte GJC.Y. ZhaoX. LiaoA.E. KrystaM.J. BertoldoD. RichaniR.B. Gilchris

In vitro survival of follicles in prepubertal ewes ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification

International audienc

Androgen signaling pathways driving reproductive and metabolic phenotypes in a PCOS mouse model

As the mechanistic basis of polycystic ovary syndrome (PCOS) remains unknown, current management relies on symptomatic treatment. Hyperandrogenism is a major PCOS characteristic and evidence supports it playing a key role in PCOS pathogenesis. Classically, androgens can act directly through the androgen receptor (AR) or, indirectly, following aromatization, via the estrogen receptor (ER). We investigated the mechanism of androgenic actions driving PCOS by comparing the capacity of non-aromatizable dihydrotestosterone (DHT) and aromatizable testosterone to induce PCOS traits in WT and Ar-knockout (ARKO) mice. DHT and testosterone induced the reproductive PCOS-like features of acyclicity and anovulation in WT females. In ARKO mice, DHT did not cause reproductive dysfunction; however, testosterone treatment induced irregular cycles and ovulatory disruption. These findings indicate that direct AR actions and indirect, likely ER, actions of androgens are important mediators of PCOS reproductive traits. DHT, but not testosterone, induced an increase in body weight, body fat, serum cholesterol and adipocyte hypertrophy in WT mice, but neither androgen induced these metabolic features in ARKO mice. These data infer that direct AR-driven mechanisms are key in driving the development of PCOS metabolic traits. Overall, these findings demonstrate that differing PCOS traits can be mediated via different steroid signaling pathways and indicate that a phenotype-based treatment approach would ensure effective targeting of the underlying mechanisms.Madeleine J Cox, Melissa C Edwards, Valentina Rodriguez Paris, Ali Aflatounian, William L Ledger, Robert B Gilchrist ... et al

A hyperandrogenic environment causes intrinsic defects that are detrimental to follicular dynamics in a PCOS mouse model

Polycystic ovary syndrome (PCOS) is a common cause of female infertility. Hyperandrogenism is both a major symptom and key diagnostic trait of PCOS; however, the direct impact of this androgen excess on ovarian dynamics is unclear. By combining a DHT-induced PCOS mouse model with an ex vivo follicle culture system, we investigated the impact of hyperandrogenism on ovarian function. Ovaries from PCOS mice exhibited the characteristic polycystic ovary morphology with numerous large cystic follicles and no corpora lutea present. Isolation and individual culture of preantral and antral follicles from PCOS mice resulted in slower growth rates during 5 days compared with the follicles isolated from control mice (P < 0.01). In contrast, preovulatory follicles from PCOS mice exhibited a significant increase in growth rate compared with controls (P < 0.01). Preantral follicles from PCOS ovaries maintained comparable follicular health as control follicles, but antral and preovulatory PCOS follicles exhibited reduced follicle health (P < 0.01) and survival rates (P < 0.01). Compared with controls, PCOS females also exhibited a poorer response to hyperstimulation (P < 0.01), impaired oocyte function evident by increased levels of reactive oxygen species (P < 0.01), and a reduction in on-time embryo development (P < 0.01). These results demonstrate that prolonged exposure to androgen excess leads to aberrant follicle development, which persists even after removal from the hyperandrogenic environment, causing perturbed follicular developmental trajectories. These findings indicate that an in vivo hyperandrogenic environment in patients with PCOS may intrinsically induce detrimental effects on follicles and oocytes.Michael J Bertoldo, Aimee S L Caldwell, Angelique H Riepsamen, Dianliang Lin, Macarena B Gonzalez, Rebecca L Robker, William L Ledger, Robert B Gilchrist, David J Handelsman, Kirsty A Walter