Michael J. Bertoldo

Seasonal variation in the ovarian function of sows

The modern domestic sow exhibits a period of impaired reproductive performance known as seasonal infertility during the late summer and early autumn months. A reduction in farrowing rate due to pregnancy loss is the most economically significant manifestation of this phenomenon. Presently, little is known of the aetiology of seasonal pregnancy loss in the pig. Recent findings represent a major advancement in the understanding of sow reproductive physiology and implicate poor oocyte developmental competence as a contributing factor to pregnancy loss during the seasonal infertility period. It has also been demonstrated that ovarian activity is depressed during the seasonal infertility period. The reduction in oocyte quality is associated with decreased levels of progesterone in follicular fluid during final oocyte maturation in vivo. The recent identification of sow-specific risk factors, such as parity for late pregnancy loss, should improve breeding herd efficiency by allowing producers to tailor management interventions and/or culling protocols that target animals identified as having a greater risk of late pregnancy loss during the seasonal infertility period.

Identification of sow-specific risk factors for late pregnancy loss during the seasonal infertility period in pigs

Reduced farrowing rates due to late pregnancy loss (LPL) is a manifestation of seasonal infertility in pigs. This study was undertaken to determine sow- and gilt-specific risk factors leading to LPL during the seasonal infertility period (January to April) in Australia. Age at first service was considered to be a major gilt-specific risk factor, whereas sow-specific factors considered included parity, prior wean-to-service interval, prior lactation length, and number of piglets weaned in the lactation period immediately preceding the mating/pregnancy event under scrutiny. Logistic regression analysis of these factors was undertaken on 13,213 animals from three farms (Farms A, B, and C). Age at first service for gilts had an effect on LPL (P<0.05) on Farm C when compared with that for Farms A and B, with those mated at approximately 220 d having the lowest rate of LPL. For older sows, parity was a factor on Farms A and C (P<0.001), with the proportion of sows with LPL increasing with increasing parity. When the data from each farm were combined and analyzed, there was a significant farm by WSI interaction, with animals from Farm C being most at-risk for LPL. Sows with shorter lactation periods (P<0.05) and smaller litters (P<0.05) at the previous lactation had a greater chance of LPL on all farms. Under the conditions of this study, we were able to identify risk factors for LPL that producers can manipulate during the seasonal infertility period to improve breeding herd productivity.

Adipokines and the Female Reproductive Tract

It is well known that adipose tissue can influence puberty, sexual maturation, and fertility in different species. Adipose tissue secretes molecules called adipokines which most likely have an endocrine effect on reproductive function. It has been revealed over the last few years that adipokines are functionally implicated at all levels of the reproductive axis including the gonad and hypothalamic-pituitary axis. Many studies have shown the presence and the role of the adipokines and their receptors in the female reproductive tract of different species. These adipokines regulate ovarian steroidogenesis, oocyte maturation, and embryo development. They are also present in the uterus and placenta where they could create a favorable environment for embryonic implantation and play a key role in maternal-fetal metabolism communication and gestation. Reproductive functions are strongly dependent on energy balance, and thereby metabolic abnormalities can lead to the development of some pathophysiologies such as polycystic ovary syndrome (PCOS). Adipokines could be a link between reproduction and energy metabolism and could partly explain some infertility related to obesity or PCOS.

AMPK: a master energy regulator for gonadal function

From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5′ AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome).

Nutritional signals and reproduction.

There is extensive evidence that nutrition influences reproductive function in various mammalian species (agricultural animals, rodents and human). However, the mechanisms underlying the relationship between nutrition, energy metabolism and reproductive function are poorly understood. This review considers nutrient sensors as a molecular link between food molecules and consequences for female and male fertility. It focuses on the roles and the molecular mechanisms of some of the relevant hormones, such as insulin and adipokines, and of energy substrates (glucose, fatty acids and amino acids), in the gonadotropic axis (central nervous system and gonads). A greater understanding of the interactions between nutrition and fertility is required for both better management of the physiological processes and the development of new molecules to prevent or cure metabolic diseases and their consequences for fertility.

Effect of metformin on the fertilizing ability of mouse spermatozoa

Numerous antioxidants have been added to cryopreservation media with varied success. The biguanide, metformin, commonly used for the treatment of type II diabetes, possesses properties impacting metabolism control that have not been yet assessed in cryopreservation protocols. The aim of this experiment was to; (i) determine the effect of metformin on fresh spermatozoa properties; and (ii) to assess positive or negative effects of metformin in post-thaw function and fertilizing capacity of mouse spermatozoa when used in cryopreservation media. The experiments have shown that the presence of metformin in fresh semen did not induce negative effects on spermatozoa quality, except a slight reduction in sperm motility at 5000μM metformin. However, when metformin was included in a cryopreservation protocol, an improvement in the fertilization rate and a reduction in the percentage of abnormal zygotes after in vitro fertilization was observed. In conclusion, metformin did not affect sperm quality at low concentrations (50μM), but its presence in the cryopreservation media could represent a benefit to improve the quality of frozen semen.

CHEMERIN (RARRES2) Decreases In Vitro Granulosa Cell Steroidogenesis and Blocks Oocyte Meiotic Progression in Bovine Species

ABSTRACT CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers—metformin and rosiglitazone—increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10−8 M) and IGF1 (10−8 M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation.

Specific deletion of AMP-activated protein kinase (α1AMPK) in murine oocytes alters junctional protein expression and mitochondrial physiology.

Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

Differences in the metabolomic signatures of porcine follicular fluid collected from environments associated with good and poor oocyte quality

The microenvironment of the developing follicle is critical to the acquisition of oocyte developmental competence, which is influenced by several factors including follicle size and season. The aim of this study was to characterise the metabolomic signatures of porcine follicular fluid (FF) collected from good and poor follicular environments, using high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Sow ovaries were collected at slaughter, 4 days after weaning, in summer and winter. The contents of small (3-4  mm) and large (5-8  mm) diameter follicles were aspirated and pooled separately for each ovary pair. Groups classified as summer-small (n=8), summer-large (n=15), winter-small (n=9) and winter-large (n=15) were analysed by 1H-NMR spectroscopy. The concentrations of 11 metabolites differed due to follicle size alone (P<0.05), including glucose, lactate, hypoxanthine and five amino acids. The concentrations of all these metabolites, except for glucose, were lower in large FF compared with small FF. Significant interaction effects of follicle size and season were found for the concentrations of glutamate, glycine, N-acetyl groups and uridine. Succinate was the only metabolite that differed in concentration due to season alone (P<0.05). The FF levels of progesterone, androstenedione and oestradiol were correlated with the concentrations of most of the metabolites examined. The results indicate that there is a distinct shift in follicular glucose metabolism as follicles increase in diameter and suggest that follicular cells may be more vulnerable to oxidative stress during the summer months. Our findings demonstrate the power of 1H-NMR spectroscopy to expand our understanding of the dynamic and complex microenvironment of the developing follicle.

Influence of heparin or the presence of cumulus cells during fertilization on the in vitro production of goat embryos

Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P<0.05) for COC that were IVF in the presence of heparin (54%) than without heparin (42%) or oocytes already denuded at collection that were IVF with or without heparin (41%; 38%; respectively). In Experiment 2, the developmental potential of oocytes denuded at collection was reduced (cleavage and blastocyst rates calculated from total oocytes: 34%; 11%, respectively) as compared to COC (77%; 59%, P<0.05). However, when equal numbers of both were mixed at the start of IVM, the rates were not significantly different to COC alone (68%; 45%), but when both were mixed equally only for IVF, the rates were reduced (57%; 40%, P<0.05). Denuded oocytes co-cultured with cumulus cells were not significantly different to intact COC (76%; 55%). The effect of adding COC during IVF to oocytes denuded after IVM was similar to adding cumulus cells to the same type of oocytes. In conclusion, both the use of heparin and the association of oocytes with cumulus cells, either detached or in intimate contact, during IVM and/or IVF significantly improve IVP of goat embryos. Furthermore, some oocytes that are already denuded at collection will develop satisfactorily to blastocysts when matured and fertilized with intact COC.