Yasmin Schuermann

Differential abundance of IGF1, bile acids, and the genes involved in their signaling in the dominant follicle microenvironment of lactating cows and nulliparous heifers

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30-50xa0days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.

Role of leptin receptors in granulosa cells during ovulation

Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4u200ah after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7u200ah post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10u200aμg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

Activated receptor tyrosine kinases in granulosa cells of ovulating follicles in mice

Successful ovulation requires the actions of gonadotropins along with those mediated by growth factors binding to their receptor tyrosine kinases (RTKs). There are several growth factors such as epidermal growth factor family ligands and interleukins that play a role during ovulation initiated by the preovulatory surge of luteinizing hormone (LH). The aim of this project was to analyze growth factor signaling pathways induced by LH in mouse granulosa cells. Immature female mice were treated with equine chorionic gonadotropin (eCG) followed 48u2009hr later by human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. We performed protein array analysis where we identified higher phosphorylation of insulin‐like growth factor 1 receptor (IGF1R), the fibroblast growth factor receptor 2 (FGFR2) and ephrin receptor B1 (EPHB1) in granulosa cells at 4u2009hr post‐hCG compared to 0u2009hr hCG (pu2009<u20090.05). We report both a significant increase in transcript abundance (pu2009<u20090.05) and the phosphorylation level (pu2009<u20090.05) of the IGF1R in granulosa cells at hCG4h. The mRNA abundance of the Fgfr2 and Ephb1 receptors remained unaltered upon hCG treatment. Nonetheless, transcript abundance of the fibroblast growth factor 2 (Fgf2) ligand was elevated at hCG4h (pu2009<u20090.01). Based on these results we conclude that the preovulatory LH surge activates signaling pathways of IGF1R through increase in the expression of the Igf1r gene in granulosa cells of ovulating follicles in mice. The LH surge also appears to activate FGFR2 IIIc and EPHB1 signaling, although further investigation is required.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes

The use of oocytes recovered from prepubertal donors for inxa0vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42xa0h), Long (LT; ≥72xa0h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5xa0mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, Pxa0<xa00.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3xa0±xa018% vs. ST 85.4xa0±xa022% vs. NT 82.2xa0±xa014%, Pxa0<xa00.05) and blastocyst rate (LT 36.7xa0±xa026% vs. ST 18.3xa0±xa015% vs. NT 16.7xa0±xa09%, Pxa0<xa00.05). Recovery rate was affected by treatment (LT 70.4xa0±xa025 vs. ST 85.4xa0±xa029 vs. NT 72.7xa0±xa023, Pxa0<xa00.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5xa0±xa011 vs. B: 14.7xa0±xa07 vs. C: 11.9xa0±xa08), the blastocyst rate increased (A: 12.8xa0±xa020% vs. B: 17.1xa0±xa021% vs. C: 21.8xa0±xa025%, Pxa0<xa00.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of inxa0vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

The fatty acid binding protein 6 gene (Fabp6) is expressed in murine granulosa cells and is involved in ovulatory response to superstimulation

The fatty acid binding protein 6 (Fabp6) is commonly regarded as a bile acid binding protein found in the distal portion of the small intestine and has been shown to be important in maintaining bile acid homeostasis. Previous studies have also reported the presence of Fabp6 in human, rat and fish ovaries, but the significance of Fabp6 in this organ is largely unknown. Therefore, we surveyed murine ovaries for Fabp6 gene expression and evaluated its role in ovarian function using mice with whole body Fabp6 deficiency. Here we show that the Fabp6 gene is expressed in granulosa and luteal cells of the mouse ovary. Treatment with gonadotropins stimulated Fabp6 gene expression in large antral follicles. The ovulation rate in response to superovulatory treatment in Fabp6-deficient mice was markedly decreased compared to wildtype (C57BL/6) mice. The results of this study suggest that expression of Fabp6 gene in granulosa cells serves an important and previously unrecognized function in fertility.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by inxa0vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6u202fmonthsu202fat 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72u202fh before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12u202fh (FSH12h), or every 8u202fh (FSH8h) or every 8u202fh untilxa0-36u202fh from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7u202f±u202f16 vs. 38.5u202f±u202f25 vs. 31.1u202f±u202f22), mean oocytes recovered (26.5u202f±u202f14 vs. 21.6u202f±u202f10 vs. 19.4u202f±u202f14) and cleavage rate (66.0u202f±u202f14 vs. 61.1u202f±u202f11 vs. 72.2u202f±u202f8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5u202f±u202f8%) compared with FSH12h (8.9u202f±u202f5%, Pu202f<u202f0.05). Oocytes from follicles of ≥5u202fmm in diameter yielded a higher rate (Pu202f<u202f0.05) of development to the blastocyst stage (13.8%) than those collected from <5u202fmm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 andu202f>u202f130 days of age, did not affect the mean number of follicles (34.2u202f±u202f15 vs. 39.3u202f±u202f26 vs. 31.6u202f±u202f25), the mean number of oocytes recovered (21.2u202f±u202f10 vs. 24.5u202f±u202f15 vs. 22.6u202f±u202f17), and the cleavage rate (68.6u202f±u202f11 vs. 61.7u202f±u202f12 vs. 70.7u202f±u202f10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9u202f±u202f6 vs. 9.5u202f±u202f8%, Pu202f<u202f0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1u202f±u202f47 vs. 91.5u202f±u202f25, Pu202f<u202f0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50u202fμM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1u202f±u202f8 vs. 18.6u202f±u202f4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

ERK1/2-dependent gene expression in the bovine ovulating follicle

Ovulation is triggered by gonadotropin surge-induced signaling cascades. To study the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in bovine ovulation, we administered the pharmacological inhibitor, PD0325901, into the preovulatory dominant follicle by intrafollicular injection. Four of five cows treated with 50u2009µM PD0325901 failed to ovulate. To uncover the molecular basis of anovulation in ERK1/2-inhibited cows, we collected granulosa and theca cells from Vehicle and PD0325901 treated follicles. Next-generation sequencing of granulosa cell RNA revealed 285 differentially expressed genes between Vehicle and PD0325901-treated granulosa cells at 6u2009h post-GnRH. Multiple inflammation-related pathways were enriched among the differentially expressed genes. The ERK1/2 dependent LH-induced genes in granulosa cells included EGR1, ADAMTS1, STAT3 and TNFAIP6. Surprisingly, PD0325901 treatment did not affect STAR expression in granulosa cells at 6u2009h post-GnRH. Granulosa cells had higher STAR protein and theca cells had higher levels of STAR mRNA in ERK1/2-inhibited follicles. Further, both granulosa and theca cells of ERK1/2-inhibited follicles had higher expression of SLC16A1, a monocarboxylate transporter, transporting substances including β-hydroxybutyrate across the plasma membrane. Taken together, ERK1/2 plays a significant role in mediating LH surge-induced gene expression in granulosa and theca cells of the ovulating follicle in cattle.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis: MICHALOVIC et al.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus–oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle‐stimulating hormone [FSH] treated and three untreated) and eight FSH‐treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real‐time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.