Semen Modulates the Expression of NGF, ABHD2, VCAN, and CTEN in the Reproductive Tract of Female Rabbits

Abstract

<jats:p>Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/β hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10–72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development.</jats:p>

Semen Modulates Inflammation and Angiogenesis in the Reproductive Tract of Female Rabbits

“…Animals were housed in the nucleus colony at the farm of the Institut de Recerca i Tecnologia Agroalimentaries (IRTA-Torre Marimon, Caldes de Montbui, Barcelona, Spain) under a controlled photoperiod and temperature [34]. Single cage equipped with plastic footrests, a feeder (restricted to 180 g/day of an all-mash pellet) and nipple-drinkers (ad libitum access to water) were used for each rabbit.…”

“…For each experimental condition, seven consecutive tissue sections from the female reproductive tracts (endocervix, distal uterus, proximal uterus, utero-tubal junction, ampulla, isthmus and infundibulum; Figure 1) were obtained after the euthanasia of the does [34]. The tissue segments were retrieved and stored in RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA) at −80 • C.…”

“…Before tissue segmentation of does included in experiment 2, the oviduct was isolated and flushed (phosphate-buffer saline supplemented with 5% fetal calf serum and 1% antibiotic-antimycotic solution) to collect the embryos, which were examined by number and developmental stage. The number of ovulated ovarian follicles (5.08 ± 2.06 follicles, mean ± standard deviation (SD)) and embryos (4.67 ± 3.14, mean ± SD) were counted for each side of the reproductive organs, as previously published elsewhere [34]. Briefly, at 24 h (2 and 4-cell), 36 h (8-cell stage), 68 h (early morula) and 72 h (morula), the embryos were collected by the method described above.…”

“…Briefly, total RNA was extracted from the tissue segments following a TRIzol-based protocol [34]. The RNA concentration of the extracts was determined from the absorbance of 260 nm with Thermo Scientific NanoDrop TM 2000 (Fisher Scientific, Gothenburg, Sweden).…”

“…High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™, Foster City, CA, USA) was used to synthesize the first-strand cDNA for quantitative polymerase chain reaction (qPCR) analyses (CFX96™; Bio-Rad Laboratories, Inc; Hercules, CA, USA). Following our previous qPCR protocol [34], the gene relative expression levels were quantified using the Pfaffl method [35]. The primer sequences, product sizes and efficiencies are shown in Table 1.…”