Ovulation of the preovulatory follicle originating from the first-wave dominant follicle leads to formation of an active corpus luteum

The objective of our study was to compare the characteristics of the corpus luteum (CL) formed after ovulation of the dominant follicle (DF) of the first follicular wave (W1) and those of the CL formed after ovulation of the DF of the second (induced) follicular wave (W2). Non-lactating Holstein cows were used for this study. In Experiment 1, cows were treated with PGF2α and GnRH on days 6 and 8 (day 0 = day of follicular wave emergence) for W1 (n = 6) and W2 (n = 6), respectively. Dominant follicles were aspirated on day 9 to quantify the amounts of mRNA (VEGF120, VEGF164, FGF-2, StAR, P450-scc and 3β-HSD) in granulosa cells (GC). In Experiment 2, the size and blood flow area of the CL formed after ovulation of the DF in W1 (W1CL; n = 6) and W2 (W2CL; n = 6) (the day of DF ovulation in W1 and W2 was day 10) were evaluated on days 12, 15, 18 and 21. The plasma P4 concentration was measured on days 10 to 21. The amounts of VEGF164, P450-scc and 3β-HSD mRNA were higher (P < 0.05) in the DF in W1, and those of VEGF120,FGF-2 and StAR mRNA tended to be higher (P < 0.1) in the DF in W1. The size of the CL was greater in the W1CL on days 15, 18 and 21. The blood flow area of the CL was greater in the W1CL on days 12 and 15. The plasma P4 concentrations were higher in the W1CL. These results indicate that the CL formed after ovulation of the DF in W1 was greater in terms of size, blood flow and plasma P4 concentration.     

Uterine blood flow during early pregnancy in ewes: interaction between the conceptus and the ovary bearing the corpus luteum

To determine if a transient increase in uterine blood flow (BF) and estradiol-17 beta (E2 beta) secretion occurs during maternal recognition of pregnancy in ewes (as previously observed for sows and cows), 40 nonpregnant (NP) ewes were assigned in equal numbers to surgery on d 9, 11, 13 or 15 postestrus (d 0 = day of estrus). For 20 NP ewes (five/day), each uterine horn (UH) was flushed with saline and uterine flushings (UF) collected. For the remaining 20 ewes, BF was determined for each UH using electromagnetic transducers, and samples of uterine arterial (UA) and uterine venous (UV) blood were obtained from each UH. After an intervening cycle, each ewe was mated, subjected to surgery on the same day postmating as during her previous nonmated cycle, and BF measurements and UA and UV samples were obtained. In addition, each UH of pregnant (P) ewes was flushed and the location of conceptuses was determined. Concentrations of E2 beta and progesterone (P4) in UA and E2 beta in UV and UF were determined by radioimmunoassay. For NP ewes, BF (ml/min) was not different for UH ipsilateral or contralateral to the ovary bearing the corpus luteum (CL), and did not differ across days, averaging 6.5 +/- .4. For P ewes, BF to UH contralateral to the ovary bearing the CL on all days and BF to UH ipsilateral to ovaries bearing CL on d 9 was similar to BF of either UH of NP ewes, averaging 6.8 +/- .6. On d 11, 13 and 15 of pregnancy, BF to UH ipsilateral to the ovary bearing CL was elevated (P less than .01) twofold (13.3 +/- .9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression pattern of HIF1alpha and vasohibins during follicle maturation and corpus luteum function in the bovine ovary

The aim of this study was to characterize expression patterns of hypoxia‐inducible factor‐1alpha (HIF1A) and vasohibin family members (VASH1 and VASH2) during different stages of ovarian function in cow. Experiment 1: Antral follicle classification occurred by follicle size and estradiol‐17beta (E2) concentration in the follicular fluid into 5 groups (<0.5, 0.5–5, 5–40, 40–180 and >180 E2 ng/ml). Experiment 2: Corpora lutea (CL) were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 (after regression) of oestrous cycle and of pregnancy (months 1–2, 3–4, 6–7, >8). Experiment 3: Cows on days 8–12 were injected with a prostaglandin F2alpha (PGF) analogue and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 hr after PGF injection. Expression of mRNA was measured by qPCR, steroid hormone concentration by EIA and localization by immunohistochemistry. HIF1A mRNA expression in our study increases significantly in follicles during final maturation. The highest HIF1A mRNA expression was detected during the early luteal phase, followed by a significant decrease afterwards. In contrast, the mRNA of vasohibins in small follicle was high, followed by a continuous and significant downregulation in preovulatory follicles. The obtained results show a remarkable inverse expression and localization pattern of HIF1A and vasohibins during different stages of ovarian function in cow. These results lead to the assumption that the examined factors are involved in the local mechanisms regulating angiogenesis and that the interactions between proangiogenic (HIF1A) and antiangiogenic (vasohibins) factors impact all stages of bovine ovary function.     

Induction of ovulation and multiple ovulations in seasonally anovulatory and ovulatory mares with an equine pituitary extract

A crude equine pituitary ethanol extract (EE) was used to induce single and miltiple ovulations in seasonally anovulatory pony mares 3-15 years of age. 12 mares were injected daily for 14 days with EE; 6 of the EE-treated mares were also treated with human chorionic gonadotropin (HCG), and 6 control mares received saline vehicle only. In a 2nd experiment designed to determine if EE treatment could induce multiple ovulations in seasonally ovulatory mares, 7 mares were treated during diestrus, 7 mares were treated beginning on Day 1 of estrus, and 7 remained untreated. The results of experiment 1 confirmed that EE treatment can induce ovulation in mares during the anovulatory season, that the timing of ovulation can be improved with HCG, and that ova from induced ovulations are fertilizable. Results of experiment 2 demonstrated that EE treatment can induce follicular activity and multiple ovulations during the ovulatory season.     

Effect of the dominant follicle aspiration before or after luteinizing hormone surge on the corpus luteum formation in the cow

Luteinizing hormone (LH) surge and follicle rupture act as trigger to start corpus luteum (CL) formation. Thus, we aimed to investigate whether a dominant follicle that has not been exposed to an LH surge can become a functional CL. For this purpose, follicular fluid from the dominant follicles (DF) of cows was aspirated before or after a GnRH-induced LH surge, and subsequent CL formation was observed. Holstein cows were divided into four groups as follows: Luteal phase, a DF was aspirated 7 days after GnRH injection; Pre-LH surge, a DF was aspirated 42 h after PGF(2alpha) injection during the mid luteal phase; Post-LH surge, a DF was aspirated 24 h after GnRH injection following PGF(2alpha); and Intact follicle, ovulation was induced by GnRH injection after PGF(2alpha). Observation of morphological changes in the aspirated follicle using color Doppler ultrasonography and blood sampling was performed on Days 0, 3, 6, and 9 (Day 0 = follicle aspiration). CL formation following DF aspiration was observed only in the Post-LH surge group. In both the Luteal phase and Pre-LH surge groups, however, none of the cows showed local blood flow at the aspirated site or CL formation. Luteal blood flow area, CL volume, and plasma progesterone concentration in the Post-LH surge group were no different from those in the Intact follicle group. The present results clearly demonstrate that rather than follicle rupture, it is the LH surge that is essential for CL formation in cows.     

Expression of melatonin and its related synthase and membrane receptors in the oestrous corpus luteum and corpus luteum verum of sheep

Melatonin is an important factor involved in regulating reproduction; it is synthesized enzymatically by the sequential action of melatonin‐synthesizing enzymes, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT), and exerts its biological functions mainly through receptor‐mediated action. To evaluate the expression of melatonin, two melatonin‐synthesizing enzymes (HIOMT and AANAT), and membrane receptors (MT1 and MT2) in oestrous corpus luteum (CL) and CL verum of sheep (Ovis aries), we performed ELISA, qRT‐PCR, western blotting and immunohistochemistry. The quantitative results showed that melatonin, HIOMT and AANAT levels in the CL verum were significantly higher than those in oestrous CL (p < 0.05), whereas MT1 and MT2 exhibited no change between the oestrous CL and CL verum (p > 0.05); moreover, the localization results showed that HIOMT, AANAT, MT1 and MT2 were mainly expressed in large luteal cells (LLCs). In summary, the above results suggested that sheep CL has potential for the synthesis of melatonin; meanwhile, they also suggested that CL is one of the targets of melatonin. These results provide not only a basis for whether sheep CL can synthesize melatonin but also provide a reference for further study on the mechanism of melatonin in the CL.     

Neural regulation of the bovine corpus luteum

The ovarian noradrenergic stimulation or noradrenaline (NA) administration directly to the ovary in cow increases ovarian oxytocin (OT) release and post-translational processing of OT synthesis within a few minutes has been established in both in vivo and in vitro studies. Furthermore, NA affects progesterone secretion and its synthesis by an increase of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta(1)- and beta(2)-receptors. Their total amount correlates with peripheral progesterone concentrations during the luteal phase and this reflects the ability of the ovary to react to beta-stimulation. On the other hand, ovarian denervation causes a decrease of steroidogenic activity in the CL, an increase of beta-receptors on luteal cells, a delay in follicular development and the disruption of cyclicity. Moreover, decrease of progesterone secretion by 20-30% was seen after brief pharmacological blockade of ovarian beta-receptors in the mid-cycle of cattle. We assume that tonic beta-stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations which could impair its function. Conversely, long-lasting increase in blood catecholamine concentrations markedly decreases the number of beta-receptors in CL, presumably due to their down-regulation. Concentrations of dopamine (DA) within the CL are highly correlated with those of NA during the estrous cycle, and are higher in the newly-formed than in the developed corpus luteum, the regressed corpus luteum or the corpus luteum of pregnant females. Bovine CL can synthesise de novo NA from DA as a precursor. Concluding, presented data indicate that noradrenergic stimulation can be an important part of mechanism supporting secretory function of CL.     

Life or death decisions in the corpus luteum

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.     

Morphological study of the corpus luteum in the rat

The cyclic related growth and regression of the corpus luteum during four consecutive oestrous cycles of the regular four-day-cycling (ie, 16 days), virgin albino Wistar rat were followed up by light and electron microscopic investigation of the ovaries. After ovulation, follicular granulosa cells differentiated into luteal cells in the newly formed corpus luteum. Based on their specific histological characteristics, four various types of corpus luteum in each stage of the oestrous cycle could be identified. As soon as the luteal cells started to degenerate, the number of fibroblasts progressively increased and apoptotic degeneration of luteal cells was initiated and became most prominent during oestrus. Complete regression of the corpus luteum was seen after 15 days. This study shows a strictly organised pattern of luteal cell growth and degeneration in the corpus luteum of the regular four-day-cycling, virgin Wistar rat. The morphological alterations may be regulated by a hormonal fluctuation.     

Roles of reactive oxygen species in the corpus luteum

Cells living under aerobic conditions always face the oxygen paradox. Oxygen is necessary for cells to maintain their lives. However, reactive oxygen species such as superoxide radicals, hydroxyl radicals and hydrogen peroxide are generated from oxygen and damage cells. Oxidative stress occurs as a consequence of the excessive production of reactive oxygen species and impaired antioxidant defense systems. Antioxidant enzymes include superoxide dismutase (SOD), which is a specific enzyme to scavenge superoxide radicals; copper‐zinc SOD, located in the cytosol and Mn‐SOD, located in the mitochondria. Both types of SOD belong to the first enzymatic step to scavenge superoxide radicals. It has been reported that a number of local factors such as cytokines, growth factors and eicosanoids are involved in the regulation of the corpus luteum (CL) function in addition to gonadotropins. Since reactive oxygen species are generated and SOD is expressed in the CL, there is a possibility that reactive oxygen species and SOD work as local regulators of the CL function. The present review reports that reactive oxygen species and their scavenging systems play important roles in the regulation of the CL function.     

The influence of the ovulation rate on ultrasonically determined ovine corpus luteum morphometry and progesterone concentrations in cyclic and early pregnant sheep

The objective of the present study was to describe morphology and function of the Corpora lutea (CL) during the oestrous cycle and early pregnancy in sheep with different ovulation rates. In total 40 Booroola. Mutton Merino crosses [heterozygous carriers (FecBFec+) and non-carriers (Fec+Fec+) of the Booroola-fecundity gene (FecB)] with ovulation rates 1 to 4 were examined. During the oestrous cycle (n = 20) and the first month of pregnancy (n = 20) blood samples were taken daily (radioimmunoassay of progesterone) and an ultrasonic ovary diagnosis was conducted. The ewes were scanned transrectally with a 7.5 MHz linear probe lying in a dorsal position. During every examination the CL could be detected. The number and the diameter of the CL were documented and the total volume of luteal tissue per ewe was calculated. The effect of the ovulation rate on CL-morphology (diameter and total volume of luteal tissue per ewe) and peripheral progesterone concentrations were assessed by one-way ANOVA. On day 6 and 7 post ovulationem in cyclic and early pregnant sheep 42% of the diagnosed CL had a cavity. On day 11 (cyclic sheep) and day 10 post ovulationem (early pregnant sheep) this number decreased to 22% (p < 0.05). Both conditions of the CL (compact or with a central cavity) are similar in function and should be regarded as appearances of the same basic process. From the third day onwards the ovulation rate influenced significantly (p < 0.05) the development of the outside diameters of the CL. However, the ovulation rate had no effect on the total volume of the luteal tissue per sheep and on the progesterone concentrations. Yet, in sheep with the ovulation rate 1 significantly lower progesterone concentrations were determined than in sheep with the ovulation rates 2 to 4. In sheep with the ovulation rates 2 to 4 the peripheral progesterone concentrations did not differ significantly. In cyclic and pregnant sheep there is a positive correlation (r = 0.75, p < 0.05) between the progesterone concentration and the total volume of luteal tissue. Considering the smaller diameters of the preovulatory follicles it seems that the development of the CL continues until a threshold-value of progesterone and/or of the total luteal tissue is exceeded. Ewes with low ovulation rates reach this threshold-value with only a few but large CL. With increasing ovulation rate the CL tend to have smaller diameters.