Corpus Luteum Function Research Articles

Sp1 regulates steroidogenic genes and LHCGR expression in primary human luteinized granulosa cells

Luteinizing hormone and human chorionic gonadotropin (hCG) bind to the luteinizing hormone/chorionic gonadotropin receptor (LHCGR). LHCGR is required to maintain corpus luteum function but the mechanisms involved in the regulation of LHCGR in human luteal cells remain incompletely understood. This study aimed to characterize the expression of LHCGR mRNA in primary human luteinized granulosa cells (hLGCs) obtained from patients undergoing in vitro fertilization and to correlate LHCGR expression with the response of hLGCs to hCG by assessing the expression of genes known to be markers of hCG actions. The results show that LHCGR expression is low in freshly isolated cells but recovers rapidly in culture and that hCG maintains LHCGR expression, suggesting a positive feedback loop. The activity of a LHCGR-LUC reporter increased in cells treated with hCG but not with follicle-stimulating hormone. Treatment with hCG also stimulated the expression of genes involved in steroidogenesis in a time-dependent manner. LHCGR promoter expression was found to be regulated by SP1, which we show is highly expressed in hLGCs. Moreover, SP1 inhibition prevented the stimulation of steroidogenic genes and the increase in LHCGR-LUC reporter activity by hCG. Finally, we provide evidence that a complex formed by SP1 and GATA4 may play a role in the maintenance of LHCGR expression. This report reveals the mechanisms involved in the regulation of the LHCGR and provides experimental data demonstrating that the proximal region of the LHCGR promoter is sufficient to drive the expression of this gene in primary hLGCs.

SFRP4 Is a Negative Regulator of Ovarian Follicle Development and Female Fertility.

WNT signaling regulates a variety of ovarian processes, including follicle development, granulosa cell (GC) proliferation and differentiation, steroidogenesis, and ovulation. The secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists. Sfrp4 expression was previously reported to be induced in ovarian GCs and cumulus cells in vivo following human chorionic gonadotropin treatment, suggesting that it may play key roles in cumulus expansion, ovulation/luteinization, and corpus luteum (CL) function. In this study, we aimed to define the physiological roles of Sfrp4 in the ovary by gene targeting. Sfrp4-null female mice were found to produce larger litters than did their wild-type littermates. Although previous studies had suggested roles of Sfrp4 in luteal cell survival, no differences in CL formation, morphology, steroidogenesis, involution, or luteal cell apoptosis were found in Sfrp4-null mice. Likewise, cumulus expansion occurred normally in Sfrp4-null mice, with minimal changes in cumulus cell gene expression. Hyperfertility in the Sfrp4-null model was ultimately attributed to decreased antral follicle atresia, leading to an enhanced ovulatory rate. Increased expression of FSH- and LH-responsive genes was found in GCs from Sfrp4-null mice, and GCs isolated from Sfrp4-null mice were found to be hyperresponsive to FSH and LH in vitro. Although Sfrp2 was found to be overexpressed in the GCs of Sfrp4-null mice (suggesting a compensatory mechanism), Sfrp2-null mice had normal fertility and ovulatory rates, and Sfrp2/4 double knockout mice did not differ from Sfrp4-null mice. Taken together, our results suggest that SFRP4 acts to attenuate GC responsiveness to gonadotropins, thereby decreasing follicle survival, ovulatory rate, and fertility.

Effect of oral administration of pioglitazone on follicular dynamics in Holstein dairy cows

This study investigated the effects of oral administration of pioglitazone (PGT), a specific and synthetic ligand of peroxisome proliferator-activated receptors gamma (PPARγ), on follicular dynamics and corpus luteum (CL) functionality in dairy cows. Cows exhibiting strong signs of estrus after 2 injections of PGF2α (given14 d apart) at d 30 postpartum (n = 28) were allotted to four groups (n = 7 cows/treatment) and orally received 6 mg PGT/kg body weight/day according to the following protocol: no PGT (control); PGT for 14 d from 7 d before expected estrus (10 d after 1st injection of PGF2α) to 7 d after observed estrus (PGT14); PGT for 21 d after observed estrus (PGT21); and PGT for 28 d, 7 d before expected estrus to 21 d after observed estrus (PGT28). During the first follicular wave, number of follicles (total and small) increased in PGT14 and PGT28 cows compared to the control group (P < 0.05). During the ovulatory wave, number of total and small follicles increased in PGT28 (P < 0.05) and PGT21 (P < 0.10) compared with PGT14 and control cows. Size of the largest follicle at first wave was greater in PGT28 (P < 0.05), PGT14 (P < 0.05) and PGT21 (P < 0.10) compared to the control cows. Maximal size of the ovulatory follicle was greater in PGT28 (P < 0.05) and PGT21 (P < 0.10) groups compared to the control group. Growth rate of the largest follicle at first wave was higher (P < 0.05) in PGT-treated cows, while growth rate of the ovulatory wave was higher in PGT28 and PGT21 groups, leading to shorter days from luteolysis to ovulation. Pioglitazone administration did not affect CL size, but increased progesterone (P4) concentration. The PGT14 and PGT28 cows had higher maximal plasma P4 concentration and shorter intervals to reach maximal plasma P4 compared to the control group. In conclusion, oral administration of PGT had some positive effects on follicular development and circulating P4 levels which may be conducive to better reproductive performance.

Using Estrous Behavior to Time Initiation of Oxytocin Administration to Prolong Luteal Function in Mares

The objective of this study was to use estrous behavior alone to determine the appropriate time for beginning an oxytocin treatment protocol for estrus suppression. We hypothesized that administration of oxytocin beginning 8 days after the onset of estrus will prolong the luteal phase in mares. Twenty-three light breed mares (aged 4–20 years) were exposed to a stallion and observed for signs of sexual receptivity. Mares not displaying signs received 250 μg of cloprostenol intramuscularly (IM) and were teased again 3–4 days later. On the day that estrous behavior was observed (Day 0), mares were randomly divided into two groups: oxytocin (n = 11): oxytocin (60 IU, IM) was administered once daily from Day 8–17; control (n = 12): did not receive treatment. Blood was collected from all mares every 4 days throughout Day 17, and every 7 days thereafter until Day 45. Serum progesterone concentrations >1.0 ng/mL were indicative of a functioning corpus luteum. Interestrus interval was defined as the period between Day 0 and the day when progesterone next reached <1.0 ng/mL. The average interestrus interval was higher for treated mares compared with control mares (32.4 ± 4.2 vs. 21.8 ± 1.5 days, respectively, P = .01). In the oxytocin group, the interestrus interval was longer than 31 days in 6 of 11 (54.5%) mares and up to 45 days in 5 of 11 mares (45.5%). We conclude that luteal maintenance beyond 30 days was attained by once-daily oxytocin administration beginning 8 days following behavioral estrus in a majority of mares.

Evaluation of a Proprietary Slow-Release Oxytocin Formulation on Corpus Luteum Function in Mares

Prolonging function of the corpus luteum (CL) is a method of suppressing estrus that relies on continued secretion of endogenous progesterone to keep mares out of heat naturally. The use of oxytocin treatment to prolong CL function is gaining increasing use, and the most common treatment protocol involves administration of 60 units of oxytocin intramuscularly (IM) once daily on days 7–14 after ovulation (eight daily treatments). Although that protocol induces prolonged CL function in ≥70% of treated mares, the need for daily administration is a drawback to its use. Therefore, the objective of this study was to evaluate the efficacy of a proprietary slow-release oxytocin formulation (SR-OT) for prolonging CL function that requires only two treatments. Mares were examined via transrectal palpation and ultrasonography to determine the day of ovulation (day 0) and then randomly assigned to a nontreated control group and an SR-OT treatment group (n = 8 mares/group). Mares in the treated group received 1.0 mL of SR-OT containing 2,400 IU oxytocin IM once on day 7 and again on day 10 after ovulation. Jugular blood samples were collected on day 0 and then every Monday, Wednesday, and Friday for 50 days for determination of the serum progesterone concentration. Mares were classified as having prolonged CL function if their progesterone concentration remained >1.0 ng/mL continuously for at least 30 days. Corpus luteum function was prolonged in 0/8 (0%) control mares and 6/8 (75%) of the SR-OT–treated mares (P < .01). The demonstrated efficacy of this two-injection, SR-OT protocol represents a 75% reduction in the number of oxytocin treatments compared with daily administration of oxytocin from day 7–14, making it a more practical treatment protocol.

Low dose human chorionic gonadotropin administration at the time of gonadotropin releasing-hormone agonist trigger versus 35\u2009h later in women at high risk of developing ovarian hyperstimulation syndrome \u2013 a prospective randomized double-blind clinical trial

BackgroundOvarian hyperstimulation syndrome remains a serious complication during in vitro fertilization cycles if high dose human chorionic gonadotropin (hCG) is used to trigger ovulation in high responder patients. Though much of this risk is mitigated with trigger using gonadotropin releasing-hormone (GnRH) agonist alone, it may result in lower birth rates. GnRH-agonist trigger and adjuvant low dose hCG has been proposed to improve birth rates, but timing of this hCG support to corpus luteum function has never been fully described. In this randomized, prospective trial, we explore differences in live birth rates and incidence of ovarian hyperstimulation syndrome (OHSS) in high-responder patients undergoing in vitro fertilization (IVF) receiving low dose hCG at the time of GnRH-agonist (dual trigger) or hCG adjuvant at the time of oocyte retrieval. Does the timing of hCG support make a difference?ResultsThirty-four subjects high-responder patients were randomized to receive low-dose hCG at the time of GnRH-agonist trigger (Group 1) and 37 received low-dose hCG at the time of oocyte retrieval (Group 2). There were no differences in the baseline characteristics and outcome of ovarian stimulation between the two groups. There were no differences in the live birth rates between Group 1 and Group 2 by intention-to-treat (14/34, 41.2% versus 21/37, 56.8%, p = 0.19) or per-protocol (14/26, 53.8% versus 19/31, 61.3%, p = 0.57) analyses. There was a slightly higher incidence of OHSS in Group 2 compared to Group 1 although the difference was not statistically significant (3/31, 9.7% versus 1/26, 3.8%). All the cases of OHSS in Group 2 were moderate while the one case of OHSS in Group 1 was mild.ConclusionsFor high responder patients receiving GnRH-agonist trigger, low dose hCG supplementation allowed high pregnancy rates after fresh embryo transfer, regardless of whether it was given at the time of trigger or at oocyte retrieval. Dual trigger may be preferable to reduce the risk of OHSS.

Dihydrotestosterone synthesis in the sheep corpus luteum and its potential mechanism in luteal regression.

Corpus luteum (CL) regression is a complex physiological process. Previous studies have shown that dihydrotestosterone (DHT) may be involved in regulating CL regression, but the mechanism is still unclear. In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in sheep CL, and investigated 5α-red1, 5α-red2, AR, and DHT levels at different luteal stages of CL (early, middle, and late phase) by immunohistochemistry, quantitative real-time polymerase chain reaction, and western blot analysis. Moreover, we cultured luteal cells from middle phase CL and treated them with different concentrations of DHT (10-10 -10 -6 M) and the AR antagonist flutamide (10 -5 M), to evaluate whether DHT is involved in the regulation of progesterone (P4) secretion and progesterone nuclear receptor (PGR) expression and whether these effects are regulated by the AR pathway. We also investigated the effects of DHT and flutamide on prostaglandin F2α (PGF2α) secretion and apoptotic gene and protein expression. Our results showed that 5α-red1, 5α-red2, and AR were expressed in the CL, and their expression and DHT levels were changed during the luteal phase. DHT was involved in mediating P4 and PGF2α secretion and PGR and apoptotic gene and protein expression. The effects of DHT on CL were at least partially regulated by the AR pathway. This study reveals the mechanism of action of DHT on sheep CL regression and lays the foundation for further exploration of androgen regulation of CL function.

Follicular fluid composition of ovulatory follicles in repeat breeder Holstein dairy cows

Objective: To examine differences in the metabolite, steroid and lipopolysaccharide of follicular fluid collected from the ovulatory follicle of Holstein repeat breeding cows, lactating cows, and virgin fertile heifers. Methods: Estrus was induced in animals possessing functional corpus luteum by intramuscular administration of prostaglandin F2 α. Six to twelve hours after detection of the standing estrus, cervical samples were collected to detect subclinical endometritis via counting neutrophils on stained smears of cervical swabs. Then, follicular fluid of ovulatory follicles and serum samples were collected from repeat breeding cows (n=11), lactating cows (n=8) and virgin fertile heifers (n=10). Sodium and potassium were measured with a flame photometer method. Urea, total protein, glucose, cholesterol and β -hydroxybutyric acid were assayed with commercial spectrophotometry kits. Chloride concentration was also measured with titration of samples against silver nitrate. Progesterone, estradiol-17 β and lipopolysaccharide concentrations were measured using enzyme-linked immuno sorbent assay kits. Results: All analysis of follicular fluid samples showed that repeat breeding and lactating cows had a mean higher lipopolysaccharide concentration than that of the virgin fertile heifers (P Conclusions: High follicular fluid concentration of lipopolysaccharide in ovulatory follicles results in the occurrence of repeat breeding syndrome in dairy cows. Further, a lower serum estradiol-17 β concentration and a higher percentage of neutrophil in the cervical secretion on the day of artificial insemination may have resulted in the occurrence of repeat breeding syndrome in dairy cows.

11 Conception rate of high-producing dairy cows at first service using prostaglandin F2α and oestradiol benzoate

We have shown that a simplified oestrus synchronization protocol using prostaglandin F2α (PG) and oestradiol benzoate (EB) resulted in a satisfactory conception rate, similar to that obtained in high-producing dairy cows with natural oestrus (Yamaguchi et al. 2018 Reprod. Fertil. Dev. 30, 147 abstr). However, we found no significant effect of PG+EB protocols on the first-service conception rates (CR) of dairy cows. This study investigated the effect of the first-service CR by using PG+EB protocols in the high-producing dairy cows that had not exhibited oestrus expression after calving. Data concerning the first-service CR were obtained for 481 Holstein cows from 4 commercial dairy herds in Hokkaido and Ibaraki, Japan, from 2014 to 2017. The average number of parity, average interval between calving and the first service, and average milk yield (at 305 days) of the cows were 2.2±1.3, 89.7±26.1 days, and 10314±1994kg, respectively. We carried out the PG+EB protocols on cows not showing evident standing heat from Days 50 to 150 postpartum regardless of the presence of a corpus luteum in the ovary. The statuses of ovaries of cows were confirmed using ultrasonography before starting the treatment. Prostaglandin F2α (25 mg; Lutalyse, Pfizer, New York, NY, USA) was injected into cows with a &amp;gt;20mm cutoff for functional corpus luteum diameter at a random stage of oestrus, and EB (1 mg; Ovahormon®, Aska Animal Health Co., Tokyo, Japan) was injected 24h later, and AI was carried out 24 to 48h after EB injection. Cows with a natural oestrus service from Days 50 to 150 postpartum were used as control. All the cows were inseminated after the onset of standing heat or removal of the tail chalk. Pregnancy diagnoses were performed using ultrasonography 30 to 45 days after AI. The CR and intervals of partum to conception were analysed using a chi-square test and ANOVA. The first-service CR (i.e. the number of pregnant cows/total number of cows in the treatment group) of the natural oestrus (n=330) and PG+EB (n=151) groups were 42.7 and 41.1%, respectively, and there were no significant differences between these 2 groups (P=0.7). The average interval between calving and the first service of the PG+EB groups was significantly longer than that of the natural oestrus groups (86.4±1.5 days v. 96.9±1.9 days; P&amp;lt;0.01). The average days open of the natural oestrus and PG+EB groups were 88.5±3.2 days and 98.1±2.3 days, respectively (P&amp;lt;0.05). These results confirmed that the first-service CR in the PG+EB and natural oestrus groups of high-producing dairy cows were similar. We suggest that conducting PG+EB protocols was effective in the high-producing dairy cows that had not exhibited oestrus expression after calving. Moreover, using PG+EB protocols could prevent the further increase in the number of days open.

Mid-Luteal 17-OH Progesterone Levels in 614 Women Undergoing IVF-Treatment and Fresh Embryo Transfer-Daytime Variation and Impact on Live Birth Rates.

Introduction: Corpus luteum (CL) produces progesterone (P4) and 17-OH progesterone (17-OH P4) during the luteal phase. Contrary to P4, 17-OH P4 is not supplied as part of the luteal phase support following IVF-treatment. Therefore, measuring endogenous serum 17-OH P4 levels may more accurately reflect the CL function compared to monitoring serum P4 concentrations.Objective: To explore the correlation between mid-luteal serum 17-OH P4 levels and live birth rates and to explore the possible daytime variations in mid-luteal serum 17-OH P4.Design: Prospective cohort study.Patients: 614 women undergoing IVF-treatment and fresh embryo transfer.Intervention: All patients had serum 17-OH P4 measured 7 days after oocyte pick-up (OPU+7). Furthermore, on OPU+7, seven patients underwent repeated blood sampling during daytime to clarify the endogenous daytime secretory pattern of 17-OH P4.Outcome measure: Live birth rate.Secondary outcome measure: Daytime variation in serum 17-OH P4 levels.Results: The highest chance of a live birth was seen with mid-luteal 17-OH P4 between 6.0 and 14.0 nmol/l. The chance of a live birth was reduced below (RD −10%, p = 0.07), but also above the optimal range for 17-OH P4 (RD −12%, p = 0.04). Patients with diminished CL-function (17-OH P4 < 6 nmol/l) displayed clinically stable 17-OH P4 values, whereas patients with 17-OH P4 levels >6 nmol/l showed random 17-OH P4 fluctuations during daytime.Conclusion: The association between 17-OH P4 and reproductive outcomes is non-linear, and the negative effect of excessive CL-secretion seems to be just as strong as the negative effect of a reduced CL-function during the peri-implantation period.

Apelina nowym regulatorem funkcji komórek jajnika

Tkanka tłuszczowa pełni aktywną funkcję wydzielniczą i uznana jest za największy gruczoł endokrynny organizmu. Adipocyty produkują i wydzielają do krwiobiegu szereg hormonów białkowych zwanych adipokinami, m.in apelinę. Hormon ten jest znanym endogennym ligandem dla receptora APJ. Ekspresję zarówno apeliny jak i jej receptora APJ wykazano w wielu tkankach, takich jak: żołądek, mózg, serce, płuca, macica czy jądra. Rolę apeliny opisano w licznych procesach fizjologicznych, jak również w patofizjologii niektórych chorób metabolicznych. Badania ostatnich lat sugerują, iż apelina reguluje również funkcje rozrodcze samic. W artykule przedstawiono aktualny stan wiedzy na temat poziomu apeliny/APJ w komórkach jajnika zarówno człowieka, jak i wielu gatunków zwierząt, a także opisano jej rolę w regulacji funkcji pęcherzyka jajnikowego i ciałka żółtego. Omówione prace jednoznacznie wskazują, na zależną od badanego gatunku ekspresję apeliny/APJ w różnych strukturach jajnika oraz rolę apeliny w procesie steroidogenezy czy apoptozy, sugerując iż jajnik jest docelową tkanką działania apeliny.

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.

Morphological study of apoptosis in granulosa cells and ovulation in a model of atresia in rat preovulatory follicles.

SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus-oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.

The Use of Power Mode Doppler Ultrasonography as a Predictive Tool of Early Pregnancy in the Mare

Human chorionic gonadotropin (hCG) has been used to induce ovulation and as a luteotrophic agent in cattle. However, the effect of hCG therapy on the functional status of the equine corpus luteum (CL) is unclear. This study aimed to characterize the hemodynamic and secretory function of early CL of mares treated with different doses of hCG at distinct stages of the estrous cycle. Mares were assigned to nine experimental groups (n = 6 mares/group) according to dose of hCG and time of treatment. A single injection of one of three different doses of hCG (750, 1,500, or 2,500 IU) was performed in one of three distinct stages of the estrous cycle: preovulatory follicle ≥35 mm, day of ovulation (D0), or 48 hours after ovulation (D2). In addition, a control group treated with NaCl 0.9% was included in the study. The end points evaluated daily from D0 to D8 were area of the CL, luteal vascularity, number of colored pixels and total pixel intensity, and concentrations of plasma progesterone (P4). No effect (P > .1) of dose or time of treatment was observed for any end point, within each day. Luteal area did not differ throughout the days (P > .1), whereas Doppler parameters and concentrations of plasma P4 presented a progressive increase (P < .05) after ovulation in all groups. Secretory function and luteal hemodynamic were not affected (P > .1) by hCG dose and time of treatment. In conclusion, hCG therapy during estrus or early diestrus, at the doses tested, did not improve P4 secretion or luteal blood flow.

Evaluation of Three Proprietary Slow-Release Oxytocin Formulations on Corpus Luteum (CL) Function in Mares

Evaluation of Three Proprietary Slow-Release Oxytocin Formulations on Corpus Luteum (CL) Function in Mares

The expression of autophagy-related proteins within the corpus luteum lifespan in pigs

Autophagy is a cellular process that involves the degradation of intracellular components. Recent studies suggested a role for autophagy in corpus luteum (CL) regression; however, a complete understanding of its contribution to CL function remains unclear. The present research using porcine CLs obtained from gilts at the early (CL1, n = 5), middle (CL2, n = 5), and late (CL3, n = 5) luteal phase of the estrous cycle aimed to assess the incidence of autophagy during CL development. The stages of collected CLs were verified through morphological analysis and intraluteal progesterone concentration. The presence of autophagosomes was assessed using transmission electron microscopy, and the expression of autophagic markers was examined at mRNA (BECN1 and Lamp1) and protein (Beclin 1, LC3-II, and Lamp 1) levels. Lamp 1 immunolocalization was also performed in luteal tissue. Double-membrane autophagosomes and autophagy-related proteins were found in all examined CLs. Interestingly, there was a greater expression of Beclin 1 (P = 0.005 and P = 0.025) and Lamp 1 (P = 0.009 and P = 0.032) protein in CL3 as compared with CL1 and CL2. In addition, the presence of autolysosomes in CL3 indicated advanced autophagy at that developmental stage. Overall, the occurrence of autophagy throughout CL development and regression suggests it has a role in the regulation of CL lifespan in pigs. In the early and mature CL, autophagy is proposed to promote luteal formation and function, whereas in the late CL, it may participate in luteal regression.

Diazinon Interrupts Ovarian Steroidogenic Acute Regulatory (StAR) Gene Transcription in Gonadotropin-Stimulated Rat Model.

Organophosphate pesticides are considered as endocrine disruptors that interfere with reproductive functions. The corpus luteum (CL) is a transient endocrine gland that produces progesterone, a crucial hormone for a successful beginning and maintenance of pregnancy. Steroidogenic acute regulatory protein (StAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to the inner organelle membranes. We investigated the effect of Diazinon (DZN), an organophosphate, on StAR mRNA expression by Sybergreen Real Time-PCR in a time-dependent manner in luteal phase. Fifty immature female Wistar rats (24-day-old) were injected with a single injection of Pregnant mare's Serum Gonadotropin (PMSG) followed by a single injection of human Chorionic Gonadotropin (hCG), 48 h later. Then, DZN was administered in a single dose (70 mg/kg bw, I.P), controls received only the vehicle, 12 h post-hCG injection. Ovaries were collected in 4 h. intervals from 8 to 24 h post-hCG injection. Then, hCG stimulation transcript levels of StAR gene were significantly altered in the hormone-stimulated rats following DZN treatment. In addition, histological study showed that the CL diameter in DZN-treated group was smaller than control group (p = 0.000). Our findings suggest that the critical step in the function of CL is disrupted by DZN and may correlates with female reproductive damage.

Detection of Genes Associated with Follicle Development Through Transcriptome Analysis of Bovine Ovarian Follicles GCs

Background: The ovarian follicle is an essential component of the reproductive process. It plays an important role in controlling the estrous cycle, determining estrous behaviour, ensuring oocyte competency and subsequent embryo survival rate, and determining both post ovulation corpus luteum function and progesterone synthesis. Traditionally, gene expression studies in the field of follicular development focus on the study of expression of candidate genes of interest. With the development of next-generation sequencing technologies, transcriptome profiling has become a powerful approach for identification of genes globally expressed in various tissues including ovarian follicles. Objective: To investigate potential differentially expressed genes associated with follicular development and gene expression profiles of bovine ovarian follicles at onset of deviation stages of a follicular wave. Method: Ovaries were removed, greatest (ODF1) and second greatest (ODF2) diameter follicles were isolated, and GCs were isolated from the two types of follicles (n=4). RNA samples of follicles were pooled within ODF1 and ODF2. The two cDNA libraries were sequenced, and mapped to the bovine RefSeq database. Gene ontology (GO) functional enrichment analysis was subjected and differentially expressed genes were analysed using visualization plug BiNGO of Cytoscape 3.0.0 software. KEGG pathways were performed and all the pathways and protein equivalent convert information of UniProt database of cattle. Twelve genes were selected randomly to verify their expression by QRT-PCR technology. Results: A total of 43,708,132 and 43,826,914 clean reads were obtained for ODF1 and ODF2, respectively. After being mapped to the bovine RefSeq database, a total of 15,519 genes were expressed (cut-off RPKM>0.5), of which 761 were highly expressed (cut-off RPKM>100) in both types of follicles. GO functional classification of these highly expressed genes revealed that many of them are involved in metabolic processes, multicellular organismal processes, and binding. Furthermore, 831 genes were identified as differentially expressed in ODF1 versus ODF2, in which 384 genes were up-regulated, and 447 genes were down-regulated. KEGG pathway analysis revealed eight differentially expressed genes in ODF1 vs. ODF2, QRT-PCR results showed that 9 of the 12 selected genes mRNA amounts were significantly greater in ODF1 than that ODF2 (P<0.05 or 0.01), which was consistent with the deep sequencing data. Conclusion: This study represents the first transcriptome investigation of bovine follicles at onset of deviation of the largest vs. second largest follicles. It provides a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle. Keywords: Follicular development, transcriptome, gene expression, GO analysis, onset of deviation.

Daytime Variation in Serum Progesterone During the Mid-Luteal Phase in Women Undergoing In Vitro Fertilization Treatment.

To investigate whether mid-luteal serum progesterone (P4) exhibits significant fluctuations during a 12-h daytime period in women undergoing in vitro fertilization (IVF) and to explore whether the extent of these fluctuations could impact the interpretation of luteal progesterone levels in a clinical setting. Explorative pilot study. Public hospital-based fertility unit. Ten women undergoing IVF treatment. Seven days after oocyte pick-up, patients underwent frequent repeated blood sampling (every 60 min for 12 h and during two of these hours, every 15 min). Serum samples were analyzed for progesterone, estradiol, and luteinizing hormone (LH). Daytime fluctuations in s-progesterone and s-estradiol. There was a significant positive correlation between median P4 levels and the magnitude of P4 variations-women with median P4 < 60 nmol/l had clinically stable P4 levels throughout the day, while patients with median P4 > 250 nmol/l exhibited periodic P4 peaks of several hundred nanomoles per liter. These endogenous P4 fluctuations were observed irrespective of the type of stimulation protocol or mode of triggering of final oocyte maturation and despite the fact that LH was under the detection limit at the time of measurement. Simultaneously, large fluctuations were seen in s-estradiol. Monitoring of early to mid-luteal P4 levels in IVF cycles may be valuable in the planning of individualized luteal phase support in the attempt to increase reproductive outcomes. The prerequisite for luteal phase monitoring is, however, that the validity of a single measured P4 value is reliable. We show for the first time, that a single P4 measurement in the low progesterone patient quite accurately reflects the corpus luteum function and that the measurement can be used to detect IVF patients with a need of additional exogenous luteal P4 administration.

Quantification of the uterine involution and dimensions, hormonal response and reproductive performance of pyometric and healthy dairy cows treated with Dinoprost

The aim of the present study was to investigate the effects of a PGF 2α analogue (Dinoprost) on involution of the puerperal uterus, especially with pyometra during 35 to 45 days postpartum in multiparous high yielding Holstein dairy cows. At days 40 ± 5 postpartum, 1250 cows were ultrasonographically examined. Fifty cows were diagnosed as pyometric. Fifty pyometric and 50 clinically healthy cows were assigned randomly to one of four groups. Groups were: 1.) Pyometric+Dinoprost cows (PD, n = 25 treated with 25 mg of Dinoprost), 2.) Pyometric+Saline cows (PS, n = 25 treated with saline), 3.) Healthy+Dinoprost cows (HD, n = 25 treated with 25 mg of Dinoprost), and 4.) Healthy+Saline cows (HS, n = 25 treated with saline). All treatments were given intramuscularly. Ultrasonography was performed at the time of examination and 7 days later to evaluate changes in uterine diameter. Treatment with Dinoprost significantly reduced gross uterine diameter in PD in comparison to the PS group in the presence or absence (P4 concentrations <1 ng/ml) of an active corpus luteum. Dinoprost treatment did not decrease gross uterine diameter in groups HD and HS. Luminal diameter was significantly smaller in the PD than in the PS group. Pregnancy rate of PD was greater than PS cows (36% vs 20%, respectively). Days to first service (110 d vs. 140 d) and open days (160 d vs. 190 d) were shorter in PD cows than PS cows. It was concluded that injection of 25 mg of Dinoprost to pyometric cows had uterotonic effects in presence or absence of functional corpus luteum. Therefore, this treatment can help uterine involution in puerperal dairy cows affected by pyometra and consequently improved reproductive performance. Keywords: Cattle, Dinoprost, pregnancy rate, pyometra, uterine health