Rami T. Kridli

The microRNAome of pregnancy: deciphering miRNA networks at the maternal-fetal interface.

MicroRNAs (miRNAs) post-transcriptionally regulate a vast network of genes by inhibiting mRNA translation. Aberrant miRNA expression profiles have been implicated in pathologies and physiological processes including pregnancy and angiogenesis. Using our established model of implantation failure and spontaneous fetal loss in pigs (Sus scrofa), 236 miRNAs were profiled and compared between 1) non-pregnant and pregnant endometrium, 2) maternal and fetal tissues, and 3) viable and growth-arrested conceptus attachment sites by microarray and Real-Time PCR. Many significant differences in miRNA expression were observed between each of the aforementioned comparisons, and several were validated by PCR. Results indicated which miRNAs were important during pregnancy, which were elevated on the maternal or fetal side of the maternal-fetal interface, and they implicated the maternal expression of miR-10a, 27a, 29c, 323, 331-5p, 339-3p, 374b-5p, and 935 in the spontaneous loss observed in pigs. Several putative mRNA targets of the miRNAs (elevated in endometrium associated with arresting conceptuses) were assessed by quantitative Real-Time PCR and were depressed, supporting their regulation by miRNAs. Finally, targets were clustered by function to obtain ranked lists of gene networks that indicated which pathways/physiological processes might be important in non-pregnant (extracellular matrix factors) versus pregnant endometrium (nuclear transcription factor regulation), maternal (blood vessel development) versus fetal (neuronal differentiation) tissue, and healthy (extracellular matrix factors) versus arresting (GRAM domain) conceptus attachment sites. Overall, we demonstrate the presence of miRNAs on both sides of the maternal-fetal interface, implicate them in spontaneous fetal loss, and present a unique glimpse into the vast microRNAome of pregnancy.

Estrus Synchronization in Sheep and Goats Using Combinations of GnRH, Progestagen and Prostaglandin F2α

The objective of this experiment was to evaluate the effect of GnRH, progestagen and prostaglandin F(2alpha) on estrus synchronization in sheep and goats. Sixty Awassi ewes and 53 Damascus does were used in the study. The experiment started at the beginning of the breeding season (June/July). The same treatments were applied to sheep and goats as follows: no treatment (CON), 14-day progestagen sponges and 600 IU equine chorionic gonadotropin (S), gonadotropin releasing hormone followed 5 days later by prostaglandin F(2alpha) (GP) and gonadotropin releasing hormone, progestagen sponges for 5 days and prostaglandin F(2alpha) on the day of sponge removal (GSP). None of the ewes in the S group lambed from mating during the induced cycle. A greater lambing rate (p < 0.05) was observed in the GSP group compared with the CON and S groups while the GP group was intermediate. The number of lambs born per lambed ewe was similar among the CON, GP and GSP groups. However, the number of lambs per exposed ewe was greater (p < 0.05) in the GSP than the remaining groups. The induced cycle kidding rate was 77% for all treatments combined. Similar kidding rate were observed among treatments. The numbers of kids born per kidded and exposed doe from mating during the induced estrus were also similar among treatments. Greater numbers of multiple births were observed in the GP and GSP than in the S group. In conclusion, a combination of GnRH, progestagen sponges and PGF(2alpha) can be effective in synchronizing estrus and improving fecundity in sheep and goats. Although the use of GnRH-PGF(2alpha) was effective, the addition of progestagen sponges at the time of GnRH administration appeared to improve reproductive parameters.

Effect of royal jelly and GnRH on the estrus synchronization and pregnancy rate in ewes using intravaginal sponges

The influence of natural royal jelly (RJ) paste and gonadotropin releasing hormone (GnRH) on reproductive responses of Awassi ewes was evaluated. In May 1998, 20 Awassi ewes (aged 2–5 years and weighing 52 kg) were treated with 40 mg fluorogestone acetate (FGA) impregnated intravaginal sponges for a period of 12 days. Half of the ewes were administered orally a total of 3 g RJ paste in 12 equal doses of 250 mg per day starting at FGA sponge insertion while the remaining half received no RJ paste. Ewes were exposed to two fertile Awassi rams from the time of FGA sponge removal (0 h, 0 day). Blood samples were drawn on alternate days from days 0 to 19. Five ewes per group (RJ and control) were randomly selected to receive a 50g GnRH injection 28 h post-sponge removal. No interactions were detected between RJ and GnRH. Incidence of estrus was greater (8/10 versus 4/10) in RJ-treated than in control ewes and intervals from 0 h to detected estrus were similar (45 ± 4.1 h versus 55 ± 6.7 h, respectively). Progesterone concentration increased on days 5–7 in all ewes and remained elevated through day 19 in 60% of the RJ-treated and 20% of the control ewes. These ewes lambed on average 149 days later and the number of lambs born was similar between the two groups of ewes. Regardless of RJ treatment, GnRH administration decreased (P < 0.05) the interval from cessation of treatment to estrus. The first rise in plasma progesterone occurred on day 5 in GnRH-treated compared with day 7 for non-GnRH-treated ewes (P < 0.05). The results demonstrate that RJ treatment in conjunction with a source of exogenous progesterone can be used to induce estrus and increase first service conception rate in sheep. Administration of GnRH reduced the interval to estrus and advanced plasma progesterone elevation.

Reproductive responses following royal jelly treatment administered orally or intramuscularly into progesterone-treated Awassi ewes

An experiment was conducted to determine whether natural royal jelly (RJ) paste administered orally or intramuscularly (i.m.) in conjunction with exogenous progesterone is associated with improved reproductive responses in ewes. Thirty 3-6-year-old Awassi ewes were randomly allocated into three (RJ-capsule, RJC; RJ-injection, RJI and control, CON) groups of 10 ewes each. All ewes were treated with intravaginal progesterone sponges for 12 days. Ewes in the RJC and RJI were administered orally or i.m. with a total of 3g of RJ given in 12 equal doses of 250 mg per ewe per day starting at the time of sponge insertion. At the time of sponge withdrawal (day 0, 0 h), ewes were exposed to three rams and checked for breeding marks at 6-h intervals for 3 days. Blood samples were collected from all ewes for analysis of progesterone concentrations. Pretreatment progesterone levels were <0.5 ng x ml(-1) in 16/30 and >1.3 ng x ml(-1) in the remaining ewes indicating luteal function and cyclicity. Similar reproductive responses and progesterone levels occurred in ewes of the RJC and RJI; therefore, data of the two groups were pooled. Following sponge insertion, progesterone levels increased rapidly and reached maximum values of 5.8+/-0.2 ng x ml(-1) within 2 days among ewes of the three groups, and then declined gradually to day 0 values of 1.6+/-0.1 and 1.9+/-0.1 ng x ml(-1) for the RJ-treated and CON ewes, respectively. The rate of progesterone decline was greater (P<0.001) in RJ-treated than in CON. Mean progesterone levels during the 12-day period were lower (P<0.001) in RJ-treated than in CON (2.8+/-0.2 ng x ml(-1) versus 3.3+/-0.2 ng x ml(-1)). Treatment with RJ resulted in greater (P<0.05) incidence of oestrus and shorter (P<0.05) intervals to onset of oestrus than CON. Based upon progesterone levels, ovulation occurred following day 0 in all ewes. Progesterone increased on day 3 in RJ-treated and on day 4 in CON ewes. Progesterone remained elevated through day 18 in 8/20 RJ-treated and 1/10 CON ewes (P=0.09). All pregnant ewes exhibited oestrus 14 h earlier (P<0.02), ovulated approximately 1 day earlier and had higher (P<0.001) luteal phase progesterone levels than non-pregnant ewes. Non-pregnant had higher (P<0.04) body weights than pregnant ewes. In conclusion, results demonstrate that both RJ treatments in conjunction with exogenous progesterone were equally capable of improving oestrus response and pregnancy rate.

Libido testing and the effect of exposing sexually naive Awassi rams to estrous ewes on sexual performance

The study was designed to test libido of Awassi rams and to detect whether their exposure to estrous ewes can improve their sexual performance. Twenty Awassi rams (yearlings and 2-year olds) were subjected to sexual performance tests. Yearlings were sexually naive at the beginning of the study. Rams were individually exposed to two estrous Awassi ewes for 30-min periods on five occasions each 2 days apart. Ram sexual behavior was recorded during each 30-min exposure. Yearling rams engaged in more (P 0.05), however, test day influenced the incidence of mounting (P<0.05) and tail-raising (P<0.01), and ejaculation rate (P<0.01). Significant correlation (P<0.01) existed between bouts of leg kicking and anogenital sniffing (r=0.4) and between tail raising and ejaculation rate (r=0.5). These results indicate that exposing sexually naive Awassi rams to estrous ewes before the breeding season may be necessary to improve their sexual performance.

Age at Puberty and Some Biological Parameters of Awassi and its First Crosses with Charollais and Romanov Rams

Abstract The aim of this experiment was to determine the age at puberty of both ram and ewe lambs of three genotypes: Awassi (A), Charollais x Awassi (CA), and Romanov x Awassi (RA) F1 crosses. Twenty, six-month old lambs (10 from each sex) from each genotype were used. Ram and ewe lambs were kept in separate groups from the beginning until the end of the experiment. Birth weight and weaning weight were recorded for all animals. Body weights and body condition scores were recorded at monthly intervals between 6.5 and 12 months of age. Blood samples were collected weekly from ewe lambs and fortnightly from ram lambs to monitor progesterone and testosterone profiles, respectively. Semen characteristics and scrotal circumferences (SC) were evaluated fortnightly. Weight at puberty in ewe and ram lambs were not significantly different among the three genotypes and ranged around 35 kg and 42 kg, respectively. In ewe lambs, age at puberty was significantly higher (P< 0.01) in A (280±11.5 d) than in RA (232±11 d) and CA (255±11.5 d). Age at puberty of ram lambs was also significantly different (P < 0.01) among the three genotypes being higher in A (243±5 d) than in CA (223±5 d) and RA (226±5 d) ram lambs. Semen parameters improved with age in all genotypes. The RA ram lambs had greater semen concentration and lower abnormal spermatozoa than the other two genotypes (P< 0.05). Results of the present study indicate that crossing Awassi ewes with either Charollais or Romanov sires tends to improve reproductive characteristics of the F1 crossbreds through advancing age at puberty in both ram and ewe lambs.

Distinct microRNA expression in endometrial lymphocytes, endometrium, and trophoblast during spontaneous porcine fetal loss

Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy.

Placentation, maternal-fetal interface, and conceptus loss in swine.

Pregnancy is a delicate yet complex physiological process that requires fine-tuning of many factors (hormones, growth factors, cytokines, and receptors) between the mother and the conceptus to ensure the survival of the conceptus(es) to term. Any disturbance in the maternal-conceptus dialog can have detrimental effects on the affected conceptus or even the outcome of pregnancy as a whole. Being a litter-bearing species, such disruptions can lead to a loss of up to 45% of the totally healthy offspring during early (periattachment) and midgestation to late gestation in pigs. Although the exact mechanism is not entirely understood, several factors have been associated with the fetal loss including but not limited to uterine capacity, placental efficiency, genetics, nutrition, and deficits in vascularization at the maternal-fetal interface. Over the years, we investigated how immune cells are recruited to the porcine maternal-fetal interface and whether they contribute to vascularization. We also delineated how cytokines, chemokines, and cytokine destabilizing factors fine-tune inflammation and whether the cytokine shift from early to midpregnancy exists at the porcine maternal-fetal interface. Finally, we evaluated the role of microRNAs in regulating immune cell recruitment and their angiogenic functions during pregnancy. Collectively our research points out that the immune-angiogenesis axis at the porcine maternal interface is significantly involved in promoting new blood vessel development, regulating inflammatory responses and ultimately contributing to pregnancy success. In this review, we summarized current knowledge on spontaneous fetal loss in swine, with special attention to the mechanisms in immune reactivity and interplay at the maternal-fetal interface.

Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk

Exosomes and microvesicles are extracellular vesicles released from cells and can contain lipids, miRNAs and proteins that affect cells at distant sites. Recently, microvesicles containing miRNA have been implicated in uterine microenvironment of pigs, a species with unique epitheliochorial (non-invasive) placentation. Here we report a novel role of conceptus-derived exosomes/microvesicles (hereafter referred to as extracellular vesicles; EVs) in embryo-endometrial cross-talk. We also demonstrate the stimulatory effects of EVs (PTr2-Exo) derived from porcine trophectoderm-cells on various biological processes including the proliferation of maternal endothelial cells (PAOEC), potentially promoting angiogenesis. Transmission immuno-electron microscopy confirmed the presence of EVs in tissue biopsies, PTr2-Exo and PAOEC-derived EVs (PAOEC-Exo). RT-PCR detected 14 select miRNAs in CD63 positive EVs in which miR-126-5P, miR-296-5P, miR-16, and miR-17-5P were the most abundant angiogenic miRNAs. Proteomic analysis revealed EV proteins that play a role in angiogenesis. In-vitro experiments, using two representative cell lines of maternal-fetal interface, demonstrated bidirectional EVs shuttling between PTr2 and PAOEC cells. Importantly, these studies support the idea that PTr2-Exo and PAOEC-Exo containing select miRNAs and proteins can be successfully delivered to recipient cells and that they may have a biological role in conceptus-endometrial cross-talk crucial for the pregnancy success.

mRNA Destabilizing Factors: Tristetraprolin Expression at the Porcine Maternal–Fetal Interface

To evaluate the expression of the tristetraprolin family and their selected targets during porcine pregnancy.