Luteal Stage Research Articles

Intra-ovarian dynamic blood flow in pseudopregnant rabbits during prostaglandin F2α-induced luteolysis.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4days) and mid-luteal (9days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40min that was followed 50min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6hr later to reach basal values after 24hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

Endometrial transcript profile of progesterone-regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis).

Progesterone (P4 ) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone-regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P4 -regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28-38th days of gestation) and stage II (48-56th days of gestation) of pregnancy (n=6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT-PCR for determining the relative mRNA expression of the P4 -regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p<0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p<0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p<0.01). These results provide molecular insights into the specific pathways involved in foeto-maternal communication during early pregnancy in buffaloes.

Analysis of differentially expressed genes and the promoters in bovine endometrium throughout estrus cycle and early pregnancy

Endometrial gene expression is primarily regulated by the ovarian steroids and pregnancy recognition factors. This study was aimed to characterize differential expression genes (DEGs) in bovine endometrium together with the analysis of their promoter region. Bovine uteri at follicular stage (FS), luteal stage (LS), and implantation stage (IS) at Day 18 of pregnancy were collected. Total RNA extracted and prepared cDNA were then subjected to high-throughput sequencing. For promoter analysis, 1kb upstream promoter region of each DEG was analyzed. The numbers of highly expressed DEGs were 496 and 597 at FS and LS, respectively. When compared the gene expression of IS with LS, 383 and 346 DEGs showed higher and lower expression at IS, respectively. It was also observed that 20-30 transcription factors (TFs) were included in each DEGs. In addition, promoter analyses estimated 150-160 TFs for each stage. DLX4 and interferon regulatory factor 4 (IRF4) at FS, and IRF5, IRF9, STAT1, and STAT2 at IS were in common to DEGs and estimated TFs, respectively. This study highlighted potential molecular mechanisms controlling endometrial function during estrus cycle and IS, which will further guide to better understand the endometrial functions in future studies.

Expression of insulin-like growth factor 1 and its receptor in preovulatory follicles and in the corpus luteum in the bitch

In the bitch, ovarian follicular and corpus luteum (CL) development and function are regulated by gonadotropins as well as local factors, the role of which is especially important during the early CL phase of relative gonadotrophic independence. We assumed that insulin-like growth factor 1 (IGF1) has a paracrine/autocrine regulatory role in ovarian follicular and luteal function in the dog. To address our hypothesis, we studied gene and protein expression of IGF1 and its receptor (IGF1R) in preovulatory follicles and in the CL of pregnant and non-pregnant dogs, and following antigestagen (aglepristone, progesterone receptor blocker) treatment in mid-gestation. Ovaries in the follicular phase were collected from five bitches. CL were collected on pregnancy Days 8–12 (pre-implantation), 18–25 (post-implantation), 35–40 (mid-gestation), at prepartum luteolysis, and 24 h and 72 h after aglepristone treatment in mid-gestation (n = 3–5 per group). From non-pregnant bitches, CL were collected on Days 5, 15, 25, 35, 45, 65 after ovulation (n = 4–5 per group). Semi-quantitative real-time (TaqMan) PCR and immunohistochemistry were applied. IGF1 immunostaining in preovulatory follicles seemed stronger in theca interna than granulosa cells. IGF1R signals appeared more intense in granulosa cells at the apical part of mural folds. In pregnant dogs, luteal IGF1 mRNA expression decreased significantly from pre-implantation to prepartum luteolysis, while IGF1R expression increased at prepartum luteolysis. Aglepristone treatment in mid-gestation had no effect on IGF1 and IGF1R mRNA levels. In non-pregnant bitches, highest IGF1 mRNA concentrations were found in the early CL and decreased by Days 45 and 65, while IGF1R expression did not change. In the CL of pregnant bitches, signals for IGF1 and IGF1R in luteal cells were strongest at pre- and post-implantation and weakest at prepartum luteolysis. IGF1 and IGF1R immunostaining was also detected in macrophages and in blood vessels. In conclusion, IGF1 may have a paracrine or autocrine role in granulosa and theca interna cells in preovulatory follicles. As IGF1 was highest represented in early luteal stages in pregnant and non-pregnant bitches, this may support a role for IGF1 in steroid synthesis, angiogenesis and cell proliferation as well as in immune function in the early canine CL. The unaffected mRNA levels after aglepristone treatment may support that IGF1 is not directly regulated by local progesterone in an auto- or paracrine manner.

The involvement of NR4A1 and NR4A2 in the regulation of the luteal function in rats

The nuclear receptor 4A (NR4A) members play important roles in cellular proliferation, differentiation and apoptosis. The current study first evaluate the expression of ovarian NR4A1 during different luteal stages in rats. Immature rats aged 28 days were treated with sequential Pregnant mare serum gonadotropin (PMSG) (D -2) / human chorionic gonadotropin (hCG) (D 0) to induce pseudopregnancy. Serum progesterone (P4) and ovarian expression of NR4A1 were detected by RIA and WB, respectively, at follicle stage (D 0), early (D 2), middle (D 7) and late (D 14 and D 20) luteal stages. To confirm the role of NR4A1 during the luteal regression, rats were treated with prostaglandin F2α analog (PGF) for 0–8 h on D 7 to detect the expressions of NR4A1 and NR4A2. RIA result showed that serum P4 reached highest level on D 7 and then declined. WB results showed that there were two types of NR4A1 (NR4A1-L and NR4A1-S) expressed in the ovary. The ovarian NR4A1-L decreased at the late luteal stage (D 20). However, the NR4A1-S increased at the late luteal stage (D 14). After PGF treatment on D 7, the expression of NR4A1-S increased which peaked at 0.5–1 h and then declined; while NR4A1-L expression did not change within 8 h. Real-time PCR results showed that the ovarian NR4A1 mRNA increased within 0.5 h, maintained high at 1 h and then declined. The NR4A2 mRNA expression exhibited a similar pattern to that of NR4A1 mRNA, though its abundance was not as high as NR4A1. IHC results revealed that NR4A1-L was expressed mainly in the cytoplasm of luteal steroidogenic cells, faintly expressed in the follicle theca cells, oocytes and the pericytes; while NR4A2 was primarily localized in the cytoplasm of luteal steroidogenic cells. In conclusion, all these results demonstrate that NR4A2 as well as NR4A1 might be involved in the luteal development and luteolysis in rats.

The expression of kisspeptin and its receptor in the domestic cat ovary and uterus in different stages of the ovarian cycle

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

The immunohistochemical localization of the fibronectin receptor (Integrin β1) and laminin in the bitch uterus during the estrous cycle

In mammals, the purpose of the estrous cycle is the successful progression of implantation and pregnancy. The endometrium undergoes several changes during the estrous cycle. The extracellular matrix proteins have a likely role in the maintenance of a healthy endometrium. In this study, we investigated the localization of the fibronectin receptor (Integrin β 1 ), a member of the integrin family, and laminin, an extracellular matrix protein, in the bitch uterus throughout the estrous cycle. In the bitch uterus, immunoreactions of similar intensity were observed for the fibronectin receptor (Integrin β 1 ) in the myometrium, perimetrium, peritoneal mesothelial cells and uterine vasculature, and for laminin in the peritoneal mesothelial cells, throughout the estrous cycle. Laminin and the fibronectin receptor (Integrin β 1 ) were found to be present at high intensity in the endometrium of the bitch uterus at the luteal stage.

Uterine Luminal Fluid Proteins in Buffalo During Follicular and Luteal Stages and Early Pregnancy

Uterine Luminal Fluid Proteins in Buffalo During Follicular and Luteal Stages and Early Pregnancy

BNIP3 expression in bovine follicle and corpus luteum.

BNIP3 (BCL2/adenovirus E1B nineteen kilodalton interacting protein-3), a member of the BCL2 family, is activated under hypoxic conditions and induces apoptosis or mitochondrial autophagy for adapting cells to hypoxia. The physiological roles of BNIP3 in the mammalian ovary are still unclear. In order to understand the role of BNIP3 in the bovine ovary, we examined its mRNA and protein expressions of BNIP3 in follicular granulosa cells and corpus luteum (CL). BNIP3 mRNA and protein expressions in granulosa cells from large follicles (>10 mm) at the follicular stage were much higher than those in small follicles (2–8 mm). BNIP3 mRNA and protein expressions in the CL peaked at the early luteal stage. In bovine granulosa cells cultured for 6 hr under hypoxia (3% O2) and normoxia (20% O2), BNIP3 mRNA expression was higher under hypoxia. These results of the present study suggest that BNIP3 has some roles in luteal formation in the bovine ovary, and that the highly expressed BNIP3 in the granulosa cells from large follicles at the follicular stage is related to the roles of BNIP3 in the luteal formation.

107 Localization of Kisspeptin Immunoreactivity in the Cat Ovary on Different Reproductive Stages

Kisspeptin (Kp) is considered one of the main regulators of the reproductive axis, exerting its effects via stimulating GnRH expression in the hypothalamus. Apart from its central localization in the hypothalamus, the presence of Kp has been reported in the ovary, with possible local function. To date, very little is known about the ovarian Kp in the domestic cat. Therefore, our aim was to investigate the presence and localization of Kp at different reproductive stages in domestic cat ovaries. Twenty ovaries were collected from free-ranging domestic cats (body weight 2.7–4.5 kg) after routine ovariohysterectomy. Reproductive stages were classified by ovarian gross morphology, vaginal cytology, and blood progesterone level. Ovarian samples were grouped into inactive (n = 6), follicular (n = 8), and luteal stages (n = 6). Tissues were fixed in 4% paraformaldehyde and processed routinely. Immunohistochemistry was performed using polyclonal rabbit Kp-10 primary antibody (AB9754; Millipore, Billerica, MA, USA) at 1:500 at 4°C overnight. Immunoreactive cells were identified by avidin-biotin-peroxidase system. Rat hypothalamic tissue was used as a positive control. Primary antibody was substituted with PBS and normal rabbit IgG as the negative and isotypic negative controls, respectively. In addition, primary antibody was incubated with metastin overnight and applied for preabsorption test. Negative, isotypic negative, and preabsorption tests showed no staining. Immunoreactive Kp was detected in the ovaries of all reproductive stages with no obvious changes in localization or intensity of staining between stages. Kisspeptin was present in the cytoplasm of oocytes, granulosa cells, and theca cells of preantral (primordial, primary, and secondary) follicles and antral follicles. Interestingly, in most follicles, Kp staining was more prominent in theca cells and oocytes compared with granulosa cells. In corpus luteum, Kp was localised in the cytoplasm of luteal cells, with more intense staining on the periphery of corpus luteum compared with the middle in 3 luteal samples, whereas the rest of the samples demonstrated homogeneous staining distribution. Apart from oocytes and steroidogenic cells, Kp was also present in the cytoplasm of cells of the ovarian surface epithelium. Our study for the first time demonstrated the presence and localization of Kp in the ovary of the domestic cats. The localization of Kp in the cat oocyte is similar to previous reports on hamsters and dogs, indicating a possible function in oocyte development. The staining in steroidogenic cells, mainly theca cells and luteal cells, is in good agreement with studies on hamsters, rats, humans, and marmosets, suggesting the possible local involvement of Kp in steroidogenesis. In addition, Kp staining in the ovarian surface epithelium suggests a possible role in the ovarian remodeling after ovulatory defects, as reported in humans and marmosets. This research was funded by the RGJ PhD program PHD/01882556; RG 7/2559.

118 Identification of KISS1 in the Domestic Cat

In the last decade, a substantial dataset has been acquired to demonstrate the critical role of kisspeptins, a family of neuropeptides derived from the KISS1 gene, in the reproduction system of mammalian and non-mammalian species. The KISS1 gene initial product can be cleaved into a 54-amino acid protein, and further degrade from the N-terminus into shorter but still active peptides (Kp-10, Kp-13, and Kp-14). The resulting peptides share a common C-terminal RF-amidated motif that leads to a strong binding with kisspeptin receptor. The Kp-10 peptide is well conserved between studied species and is thought to be essential and sufficient for the activation of kisspeptin receptor signalling pathways. In several species, the sequence of KISS1 has been obtained, and synthesised Kp-10 and Kp-54 have been successfully administered to induce ovulation. However, no studies have yet been performed to identify the KISS1 gene and its products in the domestic cat. Therefore, the aim of our work was to clone Felis catus KISS1 (fKISS1) and obtain its full sequence. Total RNA was isolated from the hypothalamus of a pubertal domestic cat collected postmortem and its quality was ensured using Agilent Bioanalyzer 2100 (RNA integrity number 8.2). Initial primers were designed based on the sequence deduced from the comparison of predicted domestic cat mRNA KISS1 sequence (XM_003999477.2) and published dog, human, cow, pig, goat, mouse, and rat mRNA KISS1 sequences. The partial mRNA fKISS1 sequence obtained here was used to design gene-specific primers for 5′ RACE and 3′ RACE PCR (GeneRacer Kit, Invitrogen, Carlsbad, CA, USA). For sequencing, 6 colonies were used per 5′ end and 3′ end of cloned fKISS1 cDNA. Additionally, the presence of KISS1 mRNA was checked via PCR in the ovaries, collected after ovariohysterectomy from domestic cats during inactive, follicular, and luteal ovarian stages. Nucleotide sequencing revealed that fKISS1 cDNA is 723 bp, and the open reading frame consists of 450 bp encoding a 149-amino acid polypeptide (kisspeptin precursor). Comparison of the overall amino acid sequence of the kisspeptin precursor revealed that domestic cat kisspeptin precursor exhibits similarity of 84, 78, 75, 68, 64, 56, and 52% to those of Hawaiian monk seal, pig, naked mole rat, human, mouse, musk shrew, and dog, respectively. The core sequence of kisspeptin, Kp-10, was highly conserved compared with other mammalian and non-mammalian species (100%, Hawaiian monk seal; 100%, mouse; 100%, pig; 100%, African clawed frog; 100%, naked mole rat; 100%, musk shrew; 90%, human; 90%, dog). Apart from hypothalamic tissue, fKISS1 has been identified in the ovaries on all stages. Obtained here results could be used for development of ovulation induction protocols in the endangered feline species. Moreover, a curious high similarity of kisspeptin sequence reported here to other induced ovulators may contribute to the research on ovulation mechanism in the domestic cat.

Relative expression of oxytocin receptor gene in buffalo endometrium in late luteal phase and pregnancy stages

ABSTRACTMolecular level information related to buffalo (Bubalus bubalis) reproduction and related genes is not present at appropriate level. If such exploration is made in the form of comparison between expression of genes is made between non-pregnant and pregnant phase, it may be helpful to aid manipulate the reproduction. Hence, the present study was carried out to reveal mRNA quantitative real time expression of oxytocin receptor (OTR) mRNA. IFN-τ is considered as the substance of maternal recognition of pregnancy and shut down the probable mechanisms which lead to luteolysis. Such mechanism includes shutting down of OTR. Therefore, relative expression of OTR was studied in endometrial tissue of three groups. The groups were non-pregnant late luteal phase, pregnancy stage I (pregnancy of <42 days) and pregnancy stage II (>42 days of pregnancy). With designed primer and GAPDH as house-keeping gene, relative mRNA expression was measured in Real-time PCR. After statistical analysis of results, the gene found to be expressed in all three stages with non-significant difference.

Expression of the vascular endothelial growth factor system (VEGF) in the porcine oviduct during the estrous cycle

The expression and localization of VEGFA and its main receptors Flt-1 and KDR is characterized in the oviduct throughout the porcine estrous cycle. Oviducts were sampled from sows at early follicular, late follicular, early luteal and late luteal stages of the estrous cycle and a segment from the mid portion of the ampulla and isthmus studied by real time RT-PCR and quantitative immunohistochemistry. The expression of the three components of the VEGF system was continuous, although differences were observed depending on the oviduct portion, the stage of the estrous cycle and the histological layer. VEGFA and KDR mRNA expressions were higher in ampulla, while Flt-1 mRNA was higher in isthmus. VEGFA protein was also higher in ampulla but Flt-1 and KDR did not show regional differences between ampulla and isthmus. While the mRNA expression of VEGFA, Flt-1 and KDR increased progressively during the luteal period, the amount of VEGFA and Flt-1 protein decreased in the same period (in isthmus only). Contrastinly, KDR protein peaked in ampulla and isthmus just before ovulation. The VEGF system was majorly located in both the secretory and ciliated cells of the oviduct epithelium, but also in the endothelium and fibroblasts of the lamina propia and the muscle fibres and vessels of the tunica muscularis. Our results are consistent with a participation of VEFG in the regulation of the dynamics of oviductal fluid secretion and the oviduct contractibility.

Metabolomics study on primary dysmenorrhea patients during the luteal regression stage based on ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

Primary dysmenorrhea (PD) is a common gynecological disorder which, while not life-threatening, severely affects the quality of life of women. Most patients with PD suffer ovarian hormone imbalances caused by uterine contraction, which results in dysmenorrhea. PD patients may also suffer from increases in estrogen levels caused by increased levels of prostaglandin synthesis and release during luteal regression and early menstruation. Although PD pathogenesis has been previously reported on, these studies only examined the menstrual period and neglected the importance of the luteal regression stage. Therefore, the present study used urine metabolomics to examine changes in endogenous substances and detect urine biomarkers for PD during luteal regression. Ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry was used to create metabolomic profiles for 36 patients with PD and 27 healthy controls. Principal component analysis and partial least squares discriminate analysis were used to investigate the metabolic alterations associated with PD. Ten biomarkers for PD were identified, including ornithine, dihydrocortisol, histidine, citrulline, sphinganine, phytosphingosine, progesterone, 17-hydroxyprogesterone, androstenedione, and 15-keto-prostaglandin F2α. The specificity and sensitivity of these biomarkers was assessed based on the area under the curve of receiver operator characteristic curves, which can be used to distinguish patients with PD from healthy controls. These results provide novel targets for the treatment of PD.

Hypoxia increases glucose transporter 1 expression in bovine corpus luteum at the early luteal stage.

A major role of the corpus luteum (CL) is to produce progesterone (P4). The CL has immature vasculature shortly after ovulation, suggesting it exists under hypoxic conditions. Hypoxia-inducible factor-1 (HIF1) induces the expression of glucose transporter 1 (GLUT1). To clarify the physiological roles of GLUT1 in bovine CL, we examined GLUT1 mRNA expression in the CL under hypoxic conditions by quantitative RT-PCR. We also measured the effects of glucose (0–25 mM) and GLUT1 inhibitors (cytochalasin B, STF-31) on P4 production in bovine luteal cells. GLUT1 mRNA expression in bovine CL was higher at the early luteal stage compared to the other later stages. Hypoxia (3% O2) increased GLUT1 mRNA expression in early luteal cells, but not in mid luteal cells. Glucose (0–25 mM) increased P4 production in early luteal cells, but not in mid luteal cells. Both GLUT1 inhibitors decreased P4 production in early and mid luteal cells. Overall, the results suggest that GLUT1 (possibly induced by hypoxic conditions in the early CL) plays a role in the establishment and development of bovine CL, especially in supporting luteal P4 synthesis at the early luteal stage.

A syntenic locus on buffalo chromosome 20: novel genomic hotspot for miRNAs involved in follicular-luteal transition.

The developmental reorganization of ovarian follicular granulosa cells (GC) during follicular maturation, ovulation, and luteinization require a well-controlled regulation of dynamic gene expression profiles. Recently, microRNAs (miRNAs) were found to be key players of ovarian follicular dynamics. The current study aimed to understand the miRNA regulatory role in follicular-luteal transition by characterizing the miRNA profile through miRNA-seq at different follicular (small, medium, and large) and luteal (early, mid, and late) stages in Indian water buffaloes, mono-ovulatory animals like humans. A total of 517 miRNAs were identified in follicular granulosa cells (GC) and corpus luteum (CL) together. Among them, 2 unique and 40 novel miRNAs were in GC; 15 unique and 45 novel miRNAs were in CL. Among the remaining 415 annotated common miRNAs between GC and CL, 43 have showed significant (p < 0.05) differential expression between GC and CL. Particularly, 39 and 4 miRNAs showed higher expression in CL and GC, respectively, with respect to each other. Genome mapping analysis revealed that 71.7% of differential miRNAs having higher expression in CL compared to GC, and 93% of the unique miRNAsin CL were mapped to a short chromosomal region of 0.7Mb (67.4 to 68.1Mb) on chromosome 21 of cows which is syntenic to the buffalo chromosome 20. Clustering of all these miRNAs at this locus suggests it as a chromosomal hotspot for miRNAs involved in follicular-luteal transition, especially for CL physiological functions.

Histological analysis of arteriovenous anastomosis-like vessels established in the corpus luteum of cows during luteolysis.

BackgroundThe mechanisms regulating the function and regression of the corpus luteum (CL) have not yet been elucidated in detail. The regressed CL of cows was previously reported to be filled with unusual vessels like arteriovenous anastomosis (AVA); however how these vessels are being established during luteolysis remains unknown.MethodsThe bovine CL at different luteal stages and regressing bovine CL induced by prostaglandin F2α (PGF) were histologically analyzed using light and electron microscopic levels. The changes in mRNA expression of genes encoding α-smooth muscle actin (SMA; Acta2) and transforming growth factor β1 (Tgfb1) in luteal tissues were analyzed by quantitative RT-PCR.ResultsAVA-like vessels appeared in the regressed CL with a diameter less than 1.5 cm in which no functional luteal cells and macrophages were observed. Epithelioid cells in the AVA-like vessel wall were immunoreactive for SMA, and the lumen of the vessels were narrow. Immunoreaction for SMA was found in the tunica media of typical arteries and arterioles, and pericytes around capillary vessel. Cells with elongated cytoplasmic processes ―resident fibroblasts expressing vimentin― distributed in the CL parenchyma without any association with blood vessels are also immunoreactive for SMA, and accumulated around arteries and arterioles during the late-luteal stage. In the regressed CL, walls of arteries and arterioles consisted of more than two layers of epithelioid cells positive for both SMA and desmin, suggesting that they are myofibroblasts transformed from fibroblasts. The percentage of the area positive for SMA and the mRNA expression of Acta2 were significantly increased in the regressed CL; however, they did not alter when a luteolytic dose of PGF was injected in vivo and collected within 24 h after the injection. On the other hand, Tgfb1, a known regulator for myofibroblast transformation, was significantly increased in PGF-induced regressing CL as well as in the CL during the late-luteal stage.ConclusionsSMA-positive myofibroblasts accumulates around the arteries and arterioles to form AVA-like vessels during luteolysis in cows. PGF indirectly regulates myofibroblast transformation through enhancing the expression of TGFβ1. These peculiar AVA-like vessels may be involved in the regulation of blood flow in the bovine CL during luteolysis.

GnRH-agonist implants suppress reproductive function and affects ovarian LHR and FSHR expression in prepubertal female cats

Effect of a GnRH-agonist (deslorelin) was studied on reproductive function and ovarian luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) expression in prepubertal female cats that were either implanted with 4.7-mg deslorelin (implanted: n = 6) or not (controls: n = 18) or ovariohysterectomized at prepubertal age (prepubertal OVH: n = 6). Body weights, fecal estradiol, and sexual behavior of implanted and control cats were monitored for 48 weeks followed by collection of ovaries and uteri. Ovaries and uteri were collected from control cats at follicular, luteal, and inactive stage (n = 6/group) and from prepubertal OVH cats at prepubertal age. Ovaries and uteri were analyzed for anatomical/histological characteristics. Ovaries were also analyzed for LHR and FSHR expression. Statistical analysis showed higher (P ≤ 0.05) body weight in control than implanted cats only during 22nd to 26th weeks of the study. Estrus was observed in control cats only. Deslorelin reduced (P ≤ 0.05) ovarian weight and number of antral follicles but did not affect endometrial thickness and gland diameter. However, myometrial thickness of implanted cats was significantly lower than control cats at follicular and luteal stage. Ovarian LHR mRNA expression was lower (P ≤ 0.05) in implanted cats than control cats at follicular stage. FSHR mRNA and LHR protein expression did not differ among the three groups. FSHR protein expression was lower (P ≤ 0.05) in prepubertal OVH cats and was not affected by deslorelin. In conclusion, deslorelin suppresses reproductive function in prepubertal female cats for at least 48 weeks possibly through a change in the ovarian mRNA expression of LHR.

Possible Contribution of Alpha2,6-Sialylation to Luteolysis in Cows by Inhibiting the Luteotropic Effects of Galectin-1.

The corpus luteum (CL) is essential for establishing pregnancy. If pregnancy does not occur during the estrous cycle, luteolysis is induced by prostaglandin (PG) F2alpha secreted from the uterus. Galectin-1, a beta-galactose-binding protein, is expressed in the functional CL of cows and increases the viability of bovine luteal steroidogenic cells (LSCs) by modifying the functions of membrane glycoproteins. The binding of galectin-1 to glycoproteins is blocked by alpha2,6-sialylation of the terminal galactose residues of glycoconjugates, which is catalyzed by a sialyltransferase (ST6Gal-I). However, the physiological role of alpha2,6-sialic acid in bovine CL is unclear. The level of alpha2,6-sialylation of the bovine CL was higher during the regressed-luteal stage than in other luteal stages. Lectin histochemistry revealed that alpha2,6-sialylated glycoconjugates were localized to luteal endothelial cells throughout the estrous cycle. In addition, alpha2,6-sialylated glycoconjugates concentrated to the membrane of LSCs during the regressed-luteal stage. PGF2alpha treatment for 72 h enhanced the expression of ST6Gal-I mRNA and the level of alpha2,6-sialylated glycoproteins in mid-LSCs. The level of alpha2,6-sialylated glycoproteins of late-stage LSCs (Days 15-17 after ovulation) was higher than that of mid-stage LSCs (Days 8-12 after ovulation), and galectin-1 increased the viability of mid-LSCs but not that of late-stage LSCs. Furthermore, galectin-1 increased the viability of late-LSCs when alpha2,6-sialic acid residues were removed by neuraminidase. The overall findings suggest that alpha2,6-sialylation stimulated by PGF2alpha contributes to luteolysis by inhibiting the luteotropic effects of galectin-1 in bovine CL.

Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx.

Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.