Prostaglandin E2 (PGE2) is a key intrafollicular mediator of ovulation in many, if not all, mammalian species. PGE2 acts at follicular cells via four distinct PGE2 receptors (PTGERs). Within the ovulatory follicle, each cell type (e.g., oocyte, cumulus granulosa cell, mural granulosa cell, theca cell, endothelial cell) expresses a different subset of the four PTGERs. Expression of a subset of PTGERs has consequences for the generation of intracellular signals and ultimately the unique functions of follicular cells that respond to PGE2. Just as the ovulatory LH surge regulates PGE2 synthesis, the LH surge also regulates expression of the four PTGERs. The pattern of expression of the four PTGERs among follicular cells before and after the LH surge forms a spatial and temporal map of PGE2 responses. Differential PTGER expression, coupled with activation of cell-specific intracellular signals, may explain how a single paracrine mediator can have pleotropic actions within the ovulatory follicle. Understanding the role of each PTGER in ovulation may point to previously unappreciated opportunities to both promote and prevent fertility.

Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Imprinted genes play an important role in placental and embryonic development. Abnormalities in their regulation can result in placental and embryonic dysplasia, leading to congenital diseases. The imprinting state, expression, and function of aquaporin-1 (Aqp1) were explored in knockout mice by imprinting analysis, real-time PCR, and immunohistochemistry. In the present study, Aqp1 was identified as a new, imprinted, and placenta-specific maternally expressed gene in the mouse. Compared with wild-type Aqp1 ^/ mice, there was significant placental and embryonic overgrowth in Aqp1^−/ (loss of maternal allele) and Aqp1^−/− mice, but not in Aqp1 ^/− (loss of paternal allele) mice at Embryonic Day (E) 12.5–E18.5. In addition, the masses of Postnatal Day 0 (P0) embryos (Aqp1^−/− and Aqp1^−/ ) were highest among the four types. In Aqp1^−/ and Aqp1^−/− mice, phenotypic analysis indicated that the number and branching of blood vessels, as well as the labyrinth area, increased significantly in placentae of E12.5–E18.5 mice. Moreover, there were abnormalities in the placental junctional zone and the labyrinthine zone at E15.5. Quantitative analysis showed that Aqp1 expression decreased significantly in the placentae of Aqp1^−/ and Aqp1^−/− mice at E15.5, and that the AQP1 protein expression signals were detected weakly in the decidual and spongioblast layers. Our results demonstrate that Aqp1 is maternally expressed in the placenta, and that its deficiency resulted in placental abnormalities in the mouse. Aqp1 may have a specific inhibitory role in mouse placental development. These results provide new insights for the treatment of diseases relating to placental and embryonic development.

Pregnancy is a complex physiological process tightly controlled by the interplay among hormones, morphogens, transcription factors, and signaling pathways. Although recent studies using genetically engineered mouse models have revealed that ligands and receptors of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) signaling pathways are essential for multiple reproductive events during pregnancy, the functional role of SMAD transcription factors, which serve as the canonical signaling platform for the TGFbeta/BMP pathways, in the oviduct and uterus is undefined. Here, we used a mouse model containing triple conditional deletion of the BMP receptor signaling Smads (Smad1 and Smad5) and Smad4, the central mediator of both TGFbeta and BMP signaling, to investigate the role of the SMADs in reproductive tract structure and function in cells from the Amhr2 lineage. Unlike the respective single- or double-knockouts, female Smad1^flox/flox Smad5^flox/flox Smad4^flox/flox Amhr2^cre/ conditional knockout (i.e., Smad1/5/4-Amhr2-cre KO) mice are sterile. We discovered that Smad1/5/4-Amhr2-cre KO females have malformed oviducts that subsequently develop oviductal diverticuli. These oviducts showed dysregulation of multiple genes essential for oviduct and smooth muscle development. In addition, uteri from Smad1/5/4-Amhr2-cre KO females exhibit multiple defects in stroma, epithelium, and smooth muscle layers and fail to assemble a closed uterine lumen upon embryo implantation, with defective uterine decidualization that led to pregnancy loss at early to mid-gestation. Taken together, our study uncovers a new role for the SMAD transcription factors in maintaining the structural and functional integrity of oviduct and uterus, required for establishment and maintenance of pregnancy.

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.

Natriuretic peptide type C (NPPC) activation of the guanylyl cyclase-linked natriuretic peptide receptor (NPR) 2 maintains oocyte meiotic arrest. Luteinizing hormone (LH)-dependent epidermal growth factor (EGF) receptor signaling elevates calcium of cumulus cells to inactivate NPR2, resulting in meiotic resumption. This study investigated the regulatory mechanism of calcium on NPR2 inactivation. In mouse ovarian follicles, LH, through the activation of EGF receptor, significantly elevated calcium levels in cumulus cells, but decreased the binding affinity of NPR2 for NPPC. In cultured cumulus-oocyte complexes, the activation of EGF receptor by EGF mobilized intracellular calcium of cumulus cells to decrease NPR2 affinity and cGMP levels, resulting in meiotic resumption. However, hormone treatments had not changed NPR2 protein levels. In addition, the removal of magnesium ions from the medium decreased the binding affinity of NPR2 for NPPC, resulting in a decrease in cGMP levels and meiotic resumption. It is concluded that magnesium ions are required to maintain functional NPR2, and that LH-dependent EGF receptor signaling mobilizes intracellular calcium of cumulus cells to reduce NPPC-NPR2 interaction that is required for meiotic resumption.

Trophoblast cells from bovine somatic cell nuclear transfer (SCNT) conceptuses express major histocompatibility complex class I (MHC-I) proteins early in gestation, and this may be one cause of the significant first-trimester embryonic mortality observed in these pregnancies. MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), and heterozygous-incompatible (n = 5) SCNT pregnancies were established. The control group consisted of eight pregnancies produced by artificial insemination. Uterine and placental samples were collected on Day 35 ± 1 of pregnancy, and expression of MHC-I, leukocyte markers, and cytokines were examined by immunohistochemistry. Trophoblast cells from all SCNT pregnancies expressed MHC-I, while trophoblast cells from age-matched control pregnancies were negative for MHC-I expression. Expression of MHC-I antigens by trophoblast cells from SCNT pregnancies was associated with lymphocytic infiltration in the endometrium. Furthermore, MHC-I-incompatible conceptuses, particularly the heterozygous-incompatible ones, induced a more pronounced lymphocytic infiltration than MHC-I-compatible conceptuses. Cells expressing cluster of differentiation (CD) 3, gamma/deltaTCR, and MHC-II were increased in the endometrium of SCNT pregnancies compared to the control group. CD4 lymphocytes were increased in MHC-I-incompatible pregnancies compared to MHC-I-compatible and control pregnancies. CD8 , FOXP3 , and natural killer cells were increased in MHC-I heterozygous-incompatible SCNT pregnancies compared to homozygous SCNT and control pregnancies.

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

Recurrent pregnancy loss (RPL), which mostly is of unknown etiology (unexplained RPL, uRPL), is defined as three or more consecutive spontaneous abortions. Some women with uRPL display a higher fraction and cytotoxicity of natural killer (NK) cells in the periphery and endometrium. Therefore, some uRPL cases have been explained by autoimmune abnormalities. The efficacy of intravenous immunoglobulin (IVIg) for uRPL has been confirmed in several clinical trials; however, its mechanism remains unknown, mainly because the abortion mechanism remains to be elucidated. In the present study, we analyzed the mechanisms of both abortion and IVIg action using a uRPL mouse model in which abortion was induced by lipopolysaccharide injection. IVIg attenuated the abortion rate in the uRPL model mice. The suppressive effect of IVIg was maximized by high dose administration early after lipopolysaccharide injection. Specifically, we discovered the presence of two distinct uterine NK (uNK) subsets: CD44^bright and CD44^mid. In uRPL model mice, we observed an increase in the number of CD44^bright uNK cells, while the CD44^mid uNK subset remained unchanged. Furthermore, when abortion was reduced by IVIg administration, the cell number of the CD44^bright uNK subset did not increase, which might allow differentiating pathological from normal uNK cells based on CD44 expression. Based on these results, we propose not only an effective administration protocol of IVIg to the uRPL model mice, but also a novel mechanism of abortion related to the increase in the CD44^bright subset and of IVIg, which suppresses the increase of the CD44^bright subset.

Endoplasmic reticulum (ER) stress results from changes in ER homeostasis and folding of proteins. ER stress initiates cellular adaptive mechanisms to rescue cell homeostasis or, if that does not work, to elicit apoptosis. We have previously shown that mouse SDF2 is sublocalized in the ER, is ubiquitously expressed, and shows strong similarities with stromal cell-derived factor (SDF) 2L1 and SDF2-like from Arabidopsis, ER proteins involved in chaperone network and protein folding. Thus, we hypothesized that SDF2 plays a role in the ER stress and unfolded protein response. In this study, we investigated the possible role of SDF2 in the human placenta. Expression of SDF2 was present throughout gestation and was expressed by several cell types. Second-trimester cytotrophoblast cells (CTBs) in the differentiation process, monitored through chorionic gonadotropin production, showed upregulation of SDF2 protein. SDF2 expression, however, was significantly diminished in placentas from neonates small for gestational age and in hypoxic in vitro conditions (P ≤ 0.001, 2% O₂), suggesting a link with cellular stress. ER stress-induced cells—CTB and BeWo—also showed SDF2 downregulation in different time points, emphasizing this relationship. SDF2 downregulation was also followed by an increase in binding immunoglobulin protein (BiP) expression, an ER protein-associated chaperone acting as a sensor for misfolded proteins and an ER stress cell survival marker. In line with this, SDF2 siRNA resulted in significant anticipation of BiP expression. Downregulation of SDF2 also interfered with C/EBP homologous protein expression, one of the highest inducible genes during ER stress. These findings suggest that SDF2 may be an important regulatory factor by which trophoblast cells can control cell survival under ER stress. In conclusion, this study identifies a novel factor with the ability to interfere with ER stress proteins, which may contribute to the understanding of ER stress associated with placental-related diseases of pregnancy.

Plasma testosterone levels are elevated in pregnant women with preeclampsia and polycystic ovaries; their offspring are at increased risk for hypertension during adult life. We tested the hypothesis that prenatal testosterone exposure induces dysregulation of the renin-angiotensin-aldosterone system, which is known to play an important role in water and electrolyte balance and blood pressure regulation. Female rats (6 mo old) prenatally exposed to testosterone were examined for adrenal expression of steroidogenic genes, telemetric blood pressure, blood volume and Na and K levels, plasma aldosterone, angiotensin II and vasopressin levels, and vascular responses to angiotensin II and arg⁸-vasopressin. The levels of Cyp11b2 (aldosterone synthase), but not the other adrenal steroidogenic genes, were decreased in testosterone females. Accordingly, plasma aldosterone levels were lower in testosterone females. Plasma volume and serum and urine Na and K levels were not significantly different between control and testosterone females; however, prenatal testosterone exposure significantly increased plasma vasopressin and angiotensin II levels and arterial pressure in adult females. In testosterone females, mesenteric artery contractile responses to angiotensin II were significantly greater, while contractile responses to vasopressin were unaffected. Angiotensin II type-1 receptor expression was increased, while angiotensin II type-2 receptor was decreased in testosterone arteries. These results suggest that prenatal testosterone exposure downregulates adrenal Cyp11b2 expression, leading to decreased plasma aldosterone levels. Elevated angiotensin II and vasopressin levels along with enhanced vascular responsiveness to angiotensin II may serve as an underlying mechanism to maintain plasma volume and Na and K levels and mediate hypertension in adult testosterone females.

The purpose of this study was to establish the time course and hemodynamic significance of de novo formed and enlarged uteroplacental arteries during pregnancy. Using x-ray microcomputed tomography (n = 4–7 placentas from 2–4 dams/gestational group), uteroplacental arterial vascular dimensions were measured at individual implantation sites. Dimensions and topology were used to compute total and vessel-specific resistances and cross-sectional areas. Diameter enlargement of the uterine artery ( 55% by Embryonic Day 5.5 [E5.5]) and preplacental radial arteries ( 30% by E8.5) was significant only in early gestation. Formation of spiral arteries (E9.5–E11.5), maternal canals, and canal branches (E11.5–E13.5) during midgestation was followed by enlargement of these vessels such that, from E9.5 to E17.5 (near term), spiral artery resistance dropped 9-fold, and canal resistance became negligible. A 12-fold increase in terminal vessel cross-sectional area was nearly sufficient to offset known increases in flow so that blood velocity entering the exchange region was predicted to increase by only 2-fold. The calculated 47% decrease in total resistance downstream of the uterine artery, determined from vascular geometry, was in accord with prior uterine blood flow data in vivo and was due to enlarging spiral artery diameters. Interestingly, radial artery resistance was unchanged after E9.5 so that radial arteries accounted for 91% of resistance and pressure drop in the uteroplacental arterial network by E17.5. These findings led us to propose functional roles for the three morphologically defined vessel types: radial arteries to reduce pressure, spiral artery enlargement to increase flow with gestation, and maternal canal elaboration and enlargement to maintain low exit velocities into the exchange region.

Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.