A. Langbeen

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles.

The Receptive Endometrial Transcriptomic Signature Indicates an Earlier Shift from Proliferation to Metabolism at Early Diestrus in the Cow.

ABSTRACT This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) −10. Devices were withdrawn and cloprostenol administered 42–60 h (LF) or 30–36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D−1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D−1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.

Unravelling the needs of singly in vitro-produced bovine embryos: from cumulus cell co-culture to semi-defined, oil-free culture conditions

Producing bovine in vitro embryos individually is a challenge as it generally leads to impaired embryo development. Earlier research optimised a single embryo in vitro production (IVP) protocol using serum, cumulus cells and oil during culture. As some of these factors are undesirable in certain circumstances, the present study investigated their necessity and possible interactions, and defined their role during single-embryo culture. Although the cumulus cell monolayer produced progesterone, it appeared not to be a key factor in supporting single-embryo development. Because in vitro culture in large medium volumes was shown to impair single-embryo development, two new oil-free culture protocols were tested. Using a 30-µL droplet of medium in 96-well plates with a small surface area resulted in comparable blastocyst rates to those obtained under oil. When serum was used, co-culture with cumulus cells seems necessary, leading to consistently high blastocyst rates. Finally, a serum-free, oil-free culture system using insulin, transferrin, selenium and BSA resulted in embryos with similar total cell numbers and apoptotic cell ratios, but blastocyst rates did not equal those obtained with serum and co-culture. This research additionally stresses the fact that specific interaction mechanisms between somatic cells and a developing in vitro embryo are far from unravelled.

Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension

The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoys 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoys medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was observed (P < 0.001), resulting in an average growth of 11.82 ± 0.81 μm (5% O2) and 11.78 ± 1.06 μm (20% O2) on Day 4 and 20.94 ± 1.24 μm (5% O2) and 19.12 ± 1.36 μm (20% O2) on Day 6 compared with Day 0. No significant differences in follicle growth rate or stage transition could be observed between 5% and 20% O2 (P > 0.05). In conclusion, after repeated NR staining, we could not find a difference between low and high oxygen tension in terms of follicle viability, stage transition, or growth. Therefore, under our culture conditions follicle dynamics are not determined by the oxygen tension in combination with quality assessment protocols using repeated NR staining.

The effect of a negative energy balance status on β-carotene availability in serum and follicular fluid of nonlactating dairy cows.

Maternal metabolic pressure due to a cows negative energy balance (NEB) has a negative effect on oocyte quality as a result of increased oxidative stress. In this study, we hypothesized that a NEB status may negatively affect the availability of β-carotene (bC, an antioxidant) in the micro-environment of the oocyte or follicular fluid (FF) and that daily bC supplementation can increase bC availability. We aimed to (1) determine the effect of a nutritionally induced NEB on bC concentrations in serum and FF as well as on the presence of bC metabolites, oxidative stress levels, and follicular growth in a nonlactating dairy cow model, and (2) investigate how this effect could be altered by dietary bC supplementation. Six multiparous nonlactating Holstein Friesian cows were subjected to 4 consecutive dietary treatments, 28 d each: (1) 1.2 × maintenance (M) or positive energy balance (PEB) without bC supplement (PEB-bC), (2) 1.2 × M with daily supplement of 2,000mg of bC comparable to the level of bC intake at grazing (PEB+bC), (3) 0.6 × M with 2,000mg of bC (NEB+bC), and (4) 0.6 × M (NEB-bC). At the end of each treatment, estrous cycles were synchronized and blood and FF of the largest follicle were sampled and analyzed for bC, retinol, α-tocopherol, free fatty acids, estradiol, and progesterone. Serum cholesterol, triglycerides, urea, insulin growth factor 1, growth hormone, total antioxidant status (TAS), and red blood cell glutathione (GSH) concentrations were determined as well. All cows lost body weight during both energy restriction periods and showed increased serum free fatty acid concentrations, illustrating a NEB. A dietary induced NEB reduced FF bC, but not plasma bC or plasma and FF retinol concentrations. However, bC and retinol concentrations drastically increased in both fluid compartments after bC supplementation. Follicular diameter was increased in supplemented PEB cows. Energy restriction reduced the TAS and red blood cell GSH, whereas daily bC supplementation could restore GSH concentrations, but not the TAS, to levels present in healthy PEB cows. In conclusion, daily bC supplementation can substantially improve bC and retinol availability in the oocytes micro-environment, irrespective of the energy balance, which may affect follicular development and oocyte quality in the presence of maternal metabolic stress. This knowledge can be of importance to optimize nutritional strategies in the dairy industry to feed for optimal oocyte quality and fertility.

Effects of parity and periconceptional metabolic state of Holstein-Friesian dams on the glucose metabolism and conformation in their newborn calves

The metabolic state of pregnant mammals influences the offsprings development and risk of metabolic disease in postnatal life. The metabolic state in a lactating dairy cow differs immensely from that in a non-lactating heifer around the time of conception, but consequences for their calves are poorly understood. The hypothesis of this study was that differences in metabolic state between non-lactating heifers and lactating cows during early pregnancy would affect insulin-dependent glucose metabolism and development in their neonatal calves. Using a mixed linear model, concentrations of glucose, IGF-I and non-esterified fatty acids (NEFAs) were compared between 13 non-lactating heifers and 16 high-yielding dairy cows in repeated blood samples obtained during the 1st month after successful insemination. Calves born from these dams were weighed and measured at birth, and subjected to intravenous glucose and insulin challenges between 7 and 14 days of age. Eight estimators of insulin-dependent glucose metabolism were determined: glucose and insulin peak concentration, area under the curve and elimination rate after glucose challenge, glucose reduction rate after insulin challenge, and quantitative insulin sensitivity check index. Effects of dam parity and calf sex on the metabolic and developmental traits were analysed in a two-way ANOVA. Compared with heifers, cows displayed lower glucose and IGF-I and higher NEFA concentrations during the 1st month after conception. However, these differences did not affect developmental traits and glucose homeostasis in their calves: birth weight, withers height, heart girth, and responses to glucose and insulin challenges in the calves were unaffected by their dams parity. In conclusion, differences in the metabolic state of heifers and cows during early gestation under field conditions could not be related to their offsprings development and glucose homeostasis.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Endometrial transcriptional profiling of a bovine fertility model by Next-Generation Sequencing

Studying the multitude of molecular networks and pathways that are potentially involved in a complex trait such as fertility requires an equally complex and broad strategy. Here, we used Next-Generation Sequencing for the characterization of the transcriptional signature of the bovine endometrial tissue. Periovulatory endocrine environments were manipulated to generate two distinctly different fertility phenotypes. Cycling, non-lactating, multiparous Nelore cows were manipulated to ovulate larger (> 13 mm; LF group; high fertility phenotype) or smaller (< 12 mm; SF group) follicles. As a result, greater proestrus estrogen concentrations, corpora lutea and early diestrus progesterone concentrations were also observed in LF group in comparison to SF group. Endometrial cell proliferation was estimated by the protein marker MKI67 on tissues collected 4 (D4) and 7 (D7) days after induction of ovulation. Total RNA extracts from D7 were sequenced and compared according to the transcriptional profile of each experimental group (LF versus SF). Functional enrichment analysis revealed that LF and SF endometria were asynchronous in regards to their phenotype manifestation. Major findings indicated an LF endometrium that was switching phenotypes earlier than the SF one. More specifically, a proliferating SF endometrium was observed on D7, whereas the LF tissue, which expressed a proliferative phenotype earlier at D4, seemed to have already shifted towards a biosynthetically and metabolically active endometrium on D7. Data on MKI67 support the transcriptomic results. RNA-Seq-derived transcriptional profile of the endometrial tissue indicated a temporal effect of the periovulatory endocrine environment, suggesting that the moment of the endometrial exposure to the ovarian steroids, E2 and P4, regulates the timing of phenotype manifestation. Gene expression profiling revealed molecules that may be targeted to elucidate ovarian steroid-dependent mechanisms that regulate endometrial tissue receptivity. Data was deposited in the SRA database from NCBI (SRA Experiment SRP051330) and are associated with the Bio-Project (PRJNA270391). An overview of the gene expression data has been deposited in NCBIs Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65450. Further assessment of the data in combination with other data sets exploring the transcriptional profile of the endometrial tissue during early diestrus may potentially identify novel molecular mechanisms and/or markers of the uterine receptivity.

Image-guided phenotyping of ovariectomized mice: altered functional connectivity, cognition, myelination and dopaminergic functionality

A large proportion of the population suffers from endocrine disruption, e.g., menopausal women, which might result in accelerated aging and a higher risk for developing cognitive disorders. Therefore, it is crucial to fully understand the impact of such disruptions on the brain to identify potential therapeutic strategies. Here, we show using resting-state functional magnetic resonance imaging that ovariectomy and consequent hypothalamus-pituitary-gonadal disruption result in the selective dysconnectivity of 2 discrete brain regions in mice. This effect coincided with cognitive deficits and an underlying pathological molecular phenotype involving an imbalance of neurodevelopmental/neurodegenerative signaling. Furthermore, this quantitative mass spectrometry proteomics-based analysis of molecular signaling patterns further identified a strong involvement of altered dopaminergic functionality (e.g., DAT and predicted upstream regulators DRD3, NR4A2), reproductive signaling (e.g., Srd5a2), rotatin expression (rttn), cellular aging (e.g., Rxfp3, Git2), myelination, and axogenesis (e.g., Nefl, Mag). With this, we have provided an improved understanding of the impact of hypothalamus-pituitary-gonadal dysfunction and highlighted the potential of using a highly translational magnetic resonance imaging technique for monitoring these effects on the brain.

Effects of vitrification on the viability of alginate encapsulated isolated bovine pre-antral follicles

PurposeIndividual follicle cryopreservation techniques, without hydrogel support, are labor-intensive and a substantial proportion of isolated follicles are lost during handling and after warming. Therefore, the viability and morphology of isolated bovine (as a model for human) pre-antral follicles after vitrification and warming, when encapsulated in alginate beads, were investigated.MethodsBovine pre-antral follicles were mechanically isolated and divided into four different groups: (1) culture in 2% alginate beads (3D system) and vitrification in beads using mesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96-well plates (2D system) and vitrification using High Security Vitrification straws® (2DVIT), (4) culture in a 2D system (2DCUL). The same vitrification and warming protocols were used for embedded (3DVIT) and non-embedded follicles (2DVIT).ResultsNo differences were observed in follicle viability between group 2DCUL and 3DCUL. Group 3DVIT showed the lowest viability (45.9%) according to calcein and neutral red staining among all groups. Group 2DVIT displayed the highest viability (87.5%) and largest percentage of follicles with a well-preserved morphology.ConclusionsOur results show that, using a vitification protocol optimized for non-embedded follicles, 2D culture is more effective in vitrifying isolated follicles. However, embedding in alginate allow to handle follicles more efficiently, i.e., without excessive manipulation and thus less labor-intensive in combination with a reduced loss of follicles during the procedure. Based on the increased work efficiency, but lower viability and higher proportion of follicles showing impaired morphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.