RhoA/ROCK signaling antagonizes bovine trophoblast stem cell self-renewal and regulates preimplantation embryo size and differentiation.

Exponential proliferation of trophoblast stem cells (TSC) is crucial in Ruminantia to maximize numerical access to caruncles, the restricted uterine sites that permit implantation. When translating systems biology of the undifferentiated bovine trophectoderm, we uncovered that inhibition of RhoA/Rock promoted self-renewing proliferation and substantially increased blastocyst size. Analysis of transcripts suppressed by Rock inhibition revealed transforming growth factor ß1 (TGFß1) as a primary upstream effector. TGFß1 treatment induced changes consistent with differentiation in bTSCs, a response that could be replicated by induced expression of the bovine ROCK2 transgene. Rocki could partially antagonize TGFß1 effects, and TGFß receptor inhibition promoted proliferation identical to Rocki, indicating an all-encompassing upstream regulation. Morphological differentiation included formation of binucleate cells and infrequent multinucleate syncytia, features we also localize in the in vivo bovine placenta. Collectively, we demonstrate a central role for TGFß1, RhoA and Rock in inducing bTSC differentiation, attenuation of which is sufficient to sustain self-renewal and proliferation linked to blastocyst size and preimplantation development. Unraveling these mechanisms augments evolutionary/comparative physiology of the trophoblast cell lineage and placental development in eutherians.

Intraovarian injection of mesenchymal stem cells improves oocyte yield and in vitro embryo production in a bovine model of fertility loss.

Valuable female cattle are continuously subject to follicular puncture (ovum pick-up - OPU). This technique is commonly used for in-vitro embryo production, but may result in ovarian lesion. Mesenchymal stem cells (MSC) ameliorate the function of injured tissues, but their use to treat ovarian lesions in cattle has not been established. We investigated whether a local injection of MSC would reduce the negative effects of repeated OPU under acute and chronic scenarios in bovines. First, we performed four OPU sessions and injected 2.5 × 106 MSCs immediately after the 4th OPU procedure (n = 5). The treated organs (right ovary) were compared to their saline-treated counterparts (left), and presented superior production of oocytes and embryos in the three following OPU sessions (P < 0.05). Then, cows with progressive fertility loss went through three OPU sessions. Animals received MSC, saline, or MSC + FSH in both ovaries after the first OPU. In the two following OPU sessions, the MSC and MSC + FSH - treated groups failed to present any significant alteration in the number of oocytes and embryos compared to saline-treated animals. Thus, MSC have beneficial effects on the fertility of OPU-lesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

Postnatal phenotype of dairy cows is altered by in vitro embryo production using reverse X-sorted semen.

Abnormal fetuses, neonates, and adult offspring derived by assisted reproductive technologies have been reported in humans and mice and have been associated with increased likelihood of certain adult diseases. To test the hypothesis that bovine females derived by assisted reproductive technologies have altered postnatal growth and adult function, a retrospective cohort study evaluated survival, growth, and production traits of offspring derived by in vitro embryo production (IVP) with conventional (IVP-conv) or reverse X-sorted semen (IVP-sexed), multiple ovulation and embryo transfer, and artificial insemination (AI) in a large dairy herd. Live calves produced by IVP were born slightly heavier compared with AI calves. In addition, IVP-sexed calves had a higher cumulative mortality from 90 to 180 d of age compared with AI offspring. Mortality of IVP-conv and multiple ovulation and embryo transfer offspring was intermediate and not different from AI or IVP-sexed offspring. The altered phenotype of offspring from IVP-sexed extended to adult milk production. Cows derived by IVP-sexed produced less milk, fat, and protein in their first lactation compared with dairy cows derived by AI. Additionally, females born to nulliparous dams had a distinct postnatal phenotype compared with offspring from parous dams even when data were restricted to offspring of surrogate females. In conclusion, procedures associated with in vitro production of embryos involving use of reverse-sorted spermatozoa for fertilization result in an alteration of embryonic programming that persists postnatally and causes an effect on milk production in adulthood. Thus, some benefits of reverse-sorted semen for genetic improvement may be offset by adverse programming events.

A single nucleotide polymorphism in COQ9 affects mitochondrial and ovarian function and fertility in Holstein cows.

A single missense mutation at position 159 of coenzyme Q9 (COQ9) (G→A; rs109301586) has been associated with genetic variation in fertility in Holstein cattle, with the A allele associated with higher fertility. COQ9 is involved in the synthesis of coenzyme COQ10, a component of the electron transport system of the mitochondria. Here we tested whether reproductive phenotype is associated with the mutation and evaluated functional consequences for cellular oxygen metabolism, body weight changes, and ovarian function. The mutation in COQ9 modifies predicted tertiary protein structure and affected mitochondrial respiration of peripheral blood mononuclear cells. The A allele was associated with low resting oxygen consumption and high electron transport system capacity. Phenotypic measurements for fertility were evaluated for up to five lactations in a population of 2273 Holstein cows. There were additive effects of the mutation (P < 0.05) in favor of the A allele for pregnancy rate, interval from calving to conception, and services per conception. There was no association of genotype with milk production or body weight changes postpartum. The mutation in COQ9 affected ovarian function; the A allele was associated with increased mitochondrial DNA copy number in oocytes, and there were overdominance effects for COQ9 expression in oocytes, follicle number, and antimullerian hormone concentrations. Overall, results show how a gene involved in mitochondrial function is associated with overall fertility, possibly in part by affecting oocyte quality.

Sex differences in response of the bovine embryo to colony-stimulating factor 2.

We tested whether gene expression of the bovine morula is modified by CSF2 in a sex-dependent manner and if sex determines the effect of CSF2 on competence of embryos to become blastocysts. Embryos were produced in vitro using X- or Y-sorted semen and treated at Day 5 of culture with 10 ng/mL bovine CSF2 or control. In experiment 1, morulae were collected at Day 6 and biological replicates (n = 8) were evaluated for transcript abundance of 90 genes by RT-qPCR using the Fluidigm Delta Gene assay. Expression of more than one-third (33 of 90) of genes examined was affected by sex. The effect of CSF2 on gene expression was modified by sex (P < 0.05) for five genes (DDX3Y/DDX3X-like, NANOG, MYF6, POU5F1 and RIPK3) and tended (P < 0.10) to be modified by sex for five other genes (DAPK1, HOXA5, PPP2R3A, PTEN and TNFSF8). In experiment 2, embryos were treated at Day 5 with control or CSF2 and blastocysts were collected at Day 7 for immunolabeling to determine the number of inner cell mass (ICM) and trophectoderm (TE) cells. CSF2 increased the percent of putative zygotes that became blastocysts for females, but did not affect the development of males. There was no effect of CSF2 or interaction of CSF2 with sex on the total number of blastomeres in blastocysts or in the number of inner cell mass or trophectoderm cells. In conclusion, CSF2 exerted divergent responses on gene expression and development of female and male embryos. These results are evidence of sexually dimorphic responses of the preimplantation embryo to this embryokine.

Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development.

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

In vivo collection of follicular fluid and granulosa cells from individual follicles of different diameters in cattle by an adapted ovum pick-up system.

BACKGROUND: Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters. METHODS: In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle. RESULTS: In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05). CONCLUSIONS: The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.