Effects of oxytocin on cloprostenol-induced luteolysis, follicular growth, ovulation and corpus luteum function in heifers

Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT-OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (1:9 mg/d) on Days 14 to 26 after estrus. All animals received 500 microg, i.m. cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OT-OT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, s.c.) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P >0.1). Duration of the estrous cycle was shortened in saline-infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >0.1). During the first 3 d of the estrous cycle following luteal regression, fewer (P <0.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P <0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatory LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis.     

Involvement of splenocytes in the control of corpus luteum function in the rat

We previously found that splenectomy caused a 1-day delay in ovulation in cycling rats. In the present study, the role of the spleen or splenocytes in corpus luteum function was investigated by comparing long-term splenectomized (SPX) rats with intact controls. When the rats were cervically stimulated on the day of proestrus, both SPX and control rats showed prolonged diestrus for 16 days associated with an increase in serum progesterone (P). However, in SPX rats, 20 alpha-dihydroprogesterone (20 alpha-OHP) concentrations were double those of controls throughout the period of pseudopregnancy. The concentration of total progestin (P plus 20 alpha-OHP) was also higher in SPX rats. All of these phenomena were normalized by injecting the animals with splenocytes. Taken together with our previous findings, these results indicate that splenocytes are involved in the control of ovarian function.     

Transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle

An ultrasound-guided transvaginal technique for corpus luteum biopsy was developed and tested in cattle. The biopsy needle set consisted of an inner needle (o.d. 1 mm) with a 20-mm long specimen notch, an outer cannula (o.d. 1.2 mm) with a cutting edge, and an automated spring-loaded handle with trigger. The biopsy needle set was inserted into the channel guide of the handle of a convex-array transvaginal ultrasound probe. The transducer was positioned in the vaginal fornix, and the ovary was manipulated transrectally against the vaginal wall and transducer face. During monitoring on the ultrasound screen, the inner needle was pushed through the vaginal wall into the corpus luteum, and the cutting cannula was fired, cutting and trapping luteal tissue in the specimen notch. Three luteal biopsies at each of Hours 0 and 4 were taken 10 d after ovulation in 6 heifers; 6 other heifers served as controls. A biopsy core was obtained in 36 of 39 attempts (92%). The tissue specimens seemed normal based on gross evaluation. The effect of biopsy on luteal function was assessed by daily ultrasound monitoring of luteal area, by assay of progesterone concentrations in blood samples obtained daily, and by the length of the interval from biopsy to ovulation. No significant differences were found for post-biopsy function for any of the 3 end points. The results indicated repeated transvaginal, ultrasound-guided biopsy of the corpus luteum in cattle is a practical procedure and may be useful for experimental and diagnostic purposes.     

Ovarian follicular and corpus luteum changes, progesterone concentrations, estrus and ovulation following estradiol benzoate/progesterone based treatment protocol in cross-bred cows

The objectives of the present study were to investigate the effects of the stage of the estrous cycle at the start of an estradiol benzoate (EB) and progesterone (P) based treatment protocol on new follicular wave emergence, subsequent estrus and ovulation. The experiment was conducted using a crossover design with each cow (five cross-bred cows) being assigned to one of three groups at 3-month intervals within a 1-year period. Estrous cycle stage in individual cows was initially synchronized with prostaglandin F(2)alpha. After detection of estrus, each cow was injected intramuscularly (i.m.) with 2 mg EB and 200 mg P (EB/P) on day 5, 12 or 17 of the estrous cycle (estrus=day 0), followed by 1 mg EB i.m. 12 days after the EB/P treatment. Ovarian ultrasonographic examinations showed that the emergence of a new follicular wave occurred after EB/P treatment in all groups and the mean interval from EB/P treatment to wave emergence did not differ among the groups (3.2-3.8 days). All cows in each group exhibited behavioral estrus and ovulated the newly formed dominant follicle. However, cows in the day-17 group exhibited estrus 1-3 days before the second EB injection. The concentrations of progesterone showed faster reduction, during the treatment period, in the day-12 and -17 groups compared to the day-5 group. These results indicate that the EB/P treatment induces an emergence of a new follicular wave, irrespective of the estrous cycle stage at the start of treatment, but the effect of EB/P protocol on estrous/ovulation synchronization is influenced by the stage of the estrous cycle.     

Conception, embryonic development and corpus luteum function in beef cattle open for two consecutive breeding seasons

High-fertility (control cows) and low-fertility (cows and heifers not pregnant after two consecutive breeding seasons — twice-open) cyclic bovine females were treated with a single injection of 1000 IU of human chrionic gonadotropin (HCG) or 100 μg of gonadotropin releasing hormone (GnRH) to enhance and/or hasten corpus luteum formation and progesterone secretion, and improve conception rate in the low-fertility females. Hormone treatments were administered to 38 parous control cows, 34 twice-open parous cows, and 27 twice-open nonparous heifers immediately after natural mating by a fertile bull. Blood samples were collected on Days 3, 6, 9, 12, and 18 after mating for determination of systemic progesterone concentrations. Pregnancy rate at necrospy approximately 33 days after mating (range 31–37) was higher in control cows (73.0%) than in twice-open cows (48.4%; P<0.05) or twice-open heifers (34.6%; P<0.01). Pregnancy rate was not affected by the HCG or GnRH treatment. The HCG treatment increased plasma progesterone concentrations in twice-open heifers but not in control or twice-open cows. Progesterone was unaffected by the GnRH treatment. Systemic progesterone concentrations were higher in control than in twice-open females but did not differ between pregnant and nonpregnant females of Days 3, 6, 9 and 12 after mating. Enhanced gonadotropin stimulation at estrus by injection of either HCG or GnRH did not increase pregnancy rate or systemic progesterone concentrations (except in HCG-treated twice-open heifers) in low- or high-fertility females. Lower pregnancy rates in twice-open females were not associated directly with the lower systemic progesterone concentrations.     

Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-alpha knockout mice

Estrogen receptor-alpha (ERalpha) knockout (ERalphaKO) female mice are infertile. Initially, they exhibit normal follicular development, but by 4-5 wk of age, they begin to develop hemorrhagic ovarian cysts. Follicles in adult ERalphaKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERalpha is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERalphaKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERalphaKO and WT mice. Gonadotropin-treated ERalphaKO females that ovulated produced 7.09 +/- 0.77 oocytes per mouse, whereas gonadotropin-treated WT female mice had 16.17 +/- 0.84 oocytes. Surprisingly, ruptured ERalphaKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERalphaKO mice had 3-fold higher concentrations of serum progesterone than did control ERalphaKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERalphaKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERalpha, although to a lesser extent than in WT mice.     

Hammond memorial lecture. New concepts of the control of corpus luteum function

Five new concepts concerning the control of corpus luteum function in the cow have been developed in recent years. Prostacyclin (PGI-2) plays a luteotrophic role. Conversely, products of the lipoxygenase pathway of arachidonic acid metabolism, particularly 5 hydroxyeicosatetraenoic acid (5-HETE), play luteolytic roles. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin; the large cells found early in the cycle (Days 2-6) are mainly of granulosa cell origin. However, a population of large cells found after Day 10 of the cycle are of theca cell origin. Oxytocin of luteal cell origin plays a role in development of the corpus luteum and possibly in its regression. The recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system, as well as the LH-cAMP system, is involved in control of progesterone synthesis in the bovine corpus luteum. Progesterone synthesis in the small theca-derived luteal cells is primarily controlled by the cAMP system. However, elevated intracellular calcium diminishes cAMP-mediated progesterone synthesis in these cells. These findings modify our current concepts of the mechanisms of control of progesterone secretion by the corpus luteum and suggest several new lines of research.     

Identification and physical mapping of genes expressed in the corpus luteum in cattle

A representational difference analysis was performed to identify genes expressed in the corpus luteum of cattle. The corpus luteum is an ovarian structure that is essential for the establishment and maintenance of pregnancy. Knowledge of gene expression and function of corpus luteum will be important to improve fertility in humans and domestic animals. Housekeeping genes were removed from the corpus luteum representation (tester) using skeletal muscle as the subtracting agent (driver). A total of 80 clones of the final subtraction product were analysed by sequencing and 11 new bovine gene sequences were identified (pBTCL1-11). The sequences were mapped to segments of 10 different chromosomes using a somatic cell hybrid panel and a radiation hybrid panel. With one exception the locations are in agreement with published comparative maps of cattle and man. Expression in corpus luteum was verified by RT-PCR for all the 11 clones.     

Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in LH-RH-treated anoestrous ewes.

Nineteen Scottish Blackface ewes were given LH-RH (3 X 30 micrograms i.v., 90-min intervals) during anoestrus when prolactin levels were elevated. Plasma levels of prolactin were suppressed with CB 154 (twice daily, i.m.) on Days -5 to 0 (N = 5), 0 to +5 (N = 5) or -5 to +5 (N = 5) around the day of LH-RH treatment (Day 0). Control animals (N = 4) received saline on Days -5 to +5. Nine animals ovulated forming corpora lutea as judged by laparoscopy on Day +7. No difference in FSH or LH levels was found between treatments and ovulations occurred equally in all treatment groups. Progesterone levels were less than ng/ml in all animals up to Day 14. It is concluded that short-term suppression of prolactin does not affect the incidence of ovulation or corpus luteum progesterone production in LH-RH-treated anoestrous ewes.     

Endocrine and local control of the primate corpus luteum

The primate corpus luteum is a transient endocrine gland that differentiates from the ovulatory follicle midway through the ovarian (menstrual) cycle. Its formation and limited lifespan is critical for fertility, as luteal-derived progesterone is the essential steroid hormone required for embryo implantation and maintenance of intra-uterine pregnancy until the placenta develops. It is well-established that LH and the LH-like hormone, CG, are the vital luteotropic hormones during the menstrual cycle and early pregnancy, respectively. Recent advances, particularly through genome analyses and cellular studies, increased our understanding of various local factors and cellular processes associated with the development, maintenance and repression of the corpus luteum. These include paracrine or autocrine factors associated with angiogenesis (e.g., VEGF), and that mediate LH/CG actions (e.g., progesterone), or counteract luteotropic effects (i.e., local luteolysis; e.g., PGF_2α). However, areas of mystery and controversy remain, particularly regarding the signals and events that initiate luteal regression in the non-fecund cycle. Novel approaches capable of gene “knockdown” or amplification”, in vivo as well as in vitro, should identify novel or underappreciated gene products that are regulated by or modulate LH/CG actions to control the functional lifespan of the primate corpus luteum. Further advances in our understanding of luteal physiology will help to improve or control fertility for purposes ranging from preservation of endangered primate species to designing novel ovary-based contraceptives and treating ovarian disorders in women.     

Morphological and functional characteristics of the dominant follicle and corpus luteum in cattle and their influence on ovarian function

Predicting the functional activity of a dominant follicle (DF) and corpus luteum (CL) might be important before starting a superovulation regime or a synchronization program. The DF and CL were characterized morphologically by using ultrasonography and were characterized functionally by estimating the estradiol-17beta/progesterone (E2/P4) ratio. Their influence on ovarian function was estimated through their ability to ovulate at different stages of development in response to PGF2alpha-application. A total of 47 Holstein Friesian (35 cows and 12 heifers) were used in two experiments. In Experiment 1, 25 animals were examined by daily transrectal palpation and ultrasonography to follow the morphological development of the DF. The status of the DF was categorized into 3 groups (A1, B1, C1). The A1 group (n=7) contained animals with DF in the growing phase or in early static growth phase for less than 3 days. Group B1 (n=13) included animals with DF in static growth phase for 3 to 4 days, while Group C1 (n=5) comprised animals with DF keeping a plateau for more than 4 days or animals with DF in the regression phase. The DF were aspirated transvaginally and the follicular fluid (FF) was analyzed for E2 and P4. In Experiment 2, 22 animals were included. As in Experiment 1, the animals were classified into three groups (A2, n=10; B2, n=5; C2, n=7). They were treated by a single dose of PGF2alpha (25 mg, i.m.) between Days 8 and 12 of the cycle. Results showed that luteolyses occurred in all animals. The DF, which were in growing or in early static growth phase < 3 days were always E2-dominant (E2 > P4) and ovulated after PGF2alpha-application in 6/8 of cases and persisted in 2 (Group A2). The DF persisting > 4 days or that had been in regression were always P4-dominant. This type of DF regressed after PGF2alpha-application (Group C2). The DF in early static growth phase for 3 to 4 days in 5/13 cases were E2-dominant and in 8/13 cases were P4-dominant. This type of DF ovulated in 3/5 cases and regressed in 2/5 cases after PGF2alpha-application (Group B2). These results suggest that the DF is morphologically and functionally defined as long as the DF is in the growing or early static growth phase (A1, A2) for at least 2 days or if the DF is in regression (C1, C2). However, when the DF is in the static growth phase for 3 or 4 days (B1, B2), their morphological and functional characteristics are different. The CL controlls ovulation in the A and C groups and plays an abettor's roll in the B-group.     

Gonadotropin-releasing hormone-induced alteration of bovine corpus luteum function

Two experiments were conducted to evaluate effects of gonadotropin-releasing hormone (GnRH) on the function of the bovine corpus luteum during the estrous cycle. In Experiment 1, 10 beef heifers were assigned randomly into two groups; each heifer served as her own control. Heifers in Group I (n = 5) were injected i.v. with vehicle (saline) on Day 2 of the cycle (Day 0 = day of estrus) followed by an i.v. injection of 100 micrograms GnRH on Day 2 of the subsequent estrous cycle. Group II (n = 5) heifers were treated similarly except injections were given on Day 10 of the estrous cycle. All heifers were bled via the jugular vein at 15 min intervals beginning 30 min prior to injection and for 3 h after injection. Blood samples were also taken on alternate days after injection through Day 16 of the cycle. Gonadotropin-releasing hormone caused a significant release of luteinizing hormone (LH) on both treatment days with the peak occurring at 15 to 30 min postinjection. Treatment with GnRH on either Day 2 or 10 caused a reduction in serum progesterone levels on Days 12, 14 and 16 of the cycle (Group I, control 3.99, 3.97; 4.07 vs. treated 2.63, 3.45, 2.87; Group II, control 3.18, 3.82, 4.13 vs. treated 2.50, 2.82, 3.17 ng/ml, respectively; common SE = 0.24 p less than 0.03). Length of the estrous cycle did not differ between groups (Group I, control 20.7 vs. treated 20.9; Group II, control 20.7 vs. treated 21.1 days, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

The variability in the interval between estrus and ovulation in cattle and its determinants

Fertility of Holstein cows has been decreasing for years and, to a lesser extent, the fertility of heifers too but more recently. A hypothesis to explain this phenomenon may be that the chronology of events leading to ovulation is different for those animals bred nowadays when compared to what was reported previously; this would result in an inappropriate time of insemination. Therefore, two experiments were designed to investigate the relationships among estrus behavior, follicular growth, hormonal events and time of ovulation in Holstein cows and heifers. In the first experiment, the onset of estrus, follicular growth, patterns of estradiol-17beta, progesterone and LH, and the time of ovulation were studied in 12 cyclic Holstein heifers that had their estrus synchronized using the Crestar method; this was done twice, 3 weeks apart. The intervals between estrus and ovulation, estrus and the LH peak, and between the LH peak and ovulation were, respectively, 38.5 h +/-3.0, 9.1 +/- 2.0 and 29.4 h +/-1.5 (mean+/- S.E.M). The variation in the interval between estrus and the LH peak explained 80.6% of the variation in the interval between estrus and ovulation. The intervals between estrus and the LH peak, and estrus and ovulation were correlated with estradiol-17beta peak value (r=-0.423, P <0.04 and r=-0.467, P<0.02, respectively). Positive correlation coefficients for the number of follicle larger than 5 mm, and negative correlation coefficients for the size of the preovulatory follicle with the intervals between estrus and LH peak, LH peak and ovulation, and estrus and ovulation suggest an ovarian control of these intervals. In respect to its role to explain the variation in the interval between estrus and ovulation, the variation in the interval between estrus and the LH peak was evaluated further in a second set of experiments utilizing 12 pubertal Holstein heifers and 35 Holstein cows. The duration of the interval between the beginning of estrus and the LH peak was longer in heifers than in cows (4.15 h versus -1.0 h; P <0.002); the variation for this interval was higher in cows than in heifers (S.E.M.= 1.2 h versus 0.8 h; P=0.01). According to the results of these studies it can be proposed that estradiol and other product(s) of ovarian origin regulate not only the duration of intervals between the onset of estrus and the LH surge but also between the LH surge and ovulation. From the results obtained in the first experiment, it may be postulated that differences observed between cows and heifers for the duration of the interval between onset of estrus and the LH surge as well as for the variation of this interval would be observed also for the interval between the onset of estrus and ovulation. Therefore, on a practical point of view, the long interval between the onset of estrus and ovulation and the high variation of this interval, especially in cows, may be a source of low fertility and should be considered when analysing reproductive disorders.     

Mutant mouse models and their contribution to our knowledge of corpus luteum development,function and regression

The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.