Local changes in blood flow within the preovulatory follicle wall and early corpus luteum in cows

Haemodynamic changes are involved in the cyclic remodelling of ovarian tissue that occurs during final follicular growth, ovulation and new corpus luteum development. The aim of this study was to characterize the real-time changes in the blood flow within the follicle wall associated with the LH surge, ovulation and corpus luteum development in cows. Normally cyclic cows with a spontaneous ovulation (n = 5) or a GnRH-induced ovulation (n = 5) were examined by transrectal colour and pulsed Doppler ultrasonography to determine the area and the time-averaged maximum velocity (TAMXV) of the blood flow within the preovulatory follicle wall and the early corpus luteum. Ultrasonographic examinations began 48 h after a luteolytic injection of PGF(2alpha) analogue was given at the mid-luteal phase of the oestrous cycle. Cows with spontaneous ovulation were scanned at 6 h intervals until ovulation occurred. Cows with GnRH-induced ovulation were scanned just before GnRH injection (0 h), thereafter at 0.5, 1, 2, 6, 12, 24 h and at 24 h intervals up to day 5. Blood samples were collected at the same time points for oestradiol, LH and progesterone determinations. Cows with both spontaneous and GnRH-induced ovulation showed a clear increase in the plasma concentration of LH (LH surge) followed by ovulation 26-34 h later. In the colour Doppler image of the preovulatory follicle, the blood flow before the LH surge was detectable only in a small area in the base of the follicle. An acute increase in the blood flow velocity (TAMXV) was detected at 0.5 h after GnRH injection, synchronously with the initiation of the LH surge. At 12 h after the LH surge, the plasma concentrations of oestradiol decreased to basal concentrations. The TAMXV remained unchanged after the initial increase until ovulation, but decreased on day 2 (12-24 h after ovulation). In the early corpus luteum, the blood flow (area and TAMXV) gradually increased in parallel with the increase in corpus luteum volume and plasma progesterone concentration from day 2 to day 5, indicating active angiogenesis and normal luteal development. Collectively, the complex structural, secretory and functional changes that take place in the ovary before ovulation are closely associated with a local increase in the blood flow within the preovulatory follicle wall. The result of the present study provides the first visual information on vascular and blood flow changes associated with ovulation and early corpus luteum development in cows. This information may be essential for future studies involving pharmacological control of blood flow and alteration of ovarian function.     

Effect of follicular aspiration just before ovulation on corpus luteum characteristics, circulating progesterone concentrations and uterine receptivity in single-ovulating and superstimulated heifers

The aim of this study was to investigate, in unstimulated and superstimulated heifers, the effect of follicle aspiration just before ovulation on corpus luteum (CL) development, circulating progesterone (P(4)) concentrations and the ability of the uterus to support embryo development. Following follicle aspiration or ovulation timed from GNRH administration, CL development was assessed by daily ultrasonography, and CL function was assessed in terms of the capacity to produce P(4) and the expression of genes involved in steroidogenesis in luteal tissue. The capacity of the uterine environment to support conceptus development was assessed following transfer and recovery of in vitro-produced embryos. Follicular aspiration just before the expected time of ovulation leads to a significant reduction in CL diameter, CL area and area of luteal tissue. This was associated with a decrease in circulating P(4) in both unstimulated and superstimulated heifers. Follicle aspiration leads to a reduction in conceptus length and area on day 14 in unstimulated heifers only. Follicle aspiration leads to a reduction in the expression of LHCGR in luteal tissue from unstimulated heifers compared with those in which the CL formed after ovulation. Superstimulation significantly reduced the expression of STAR in luteal tissue in both ovulated and follicle-aspirated heifers. In conclusion, in stimulated and unstimulated heifers, aspiration of the preovulatory dominant follicle(s) just before expected ovulation interferes with the subsequent formation and function of the CL, in terms of size and P(4) output and this, in turn, is associated with a reduced capacity of the uterus to support conceptus elongation in unstimulated heifers.     

Genetic merit for fertility traits in Holstein cows: II. Ovarian follicular and corpus luteum dynamics, reproductive hormones, and estrus behavior

The objective of this study was to characterize the estrous cycle of cows with similar proportions of Holstein genetics, similar genetic merit for milk production traits, but with good (Fert+) or poor (Fert-) genetic merit for fertility traits. In total, 37 lactating cows were enrolled on a protocol to synchronize estrus. Nineteen Fert+ and 12 Fert- cows that successfully ovulated a dominant follicle and established a corpus luteum underwent daily transrectal ultrasonography. Blood sampling was carried out at 8-h intervals from d 0 to 6 and from d 15 to ovulation, and once daily from d 7 to 15. Blood samples were analyzed for progesterone, estradiol, follicle stimulating hormone, and luteinizing hormone. Estrus behavior was recorded using neck activity collars and mounting pads. The Fert+ cows tended to have fewer follicular waves (2.2 vs. 2.7) and had a shorter estrous cycle (21.0 vs. 25.1 d) than Fert- cows. We observed no effect of genotype on day of first-wave emergence or day of first-wave dominant follicle peak diameter, but the peak diameter of the first-wave dominant follicle tended to be larger in Fert- cows. During the first 13 d of the cycle, Fert+ cows developed a corpus luteum that was 16% larger than that in Fert- cows. Circulating progesterone concentrations were 34% greater in Fert+ than in Fert- cows (5.15 vs. 3.84ng/mL, respectively) from d 5 to 13. During the final follicular wave, the interval from preovulatory follicle emergence to ovulation and the interval from preovulatory follicle dominance to ovulation were similar in both genotypes. Maximum preovulatory follicle diameter was larger in Fert+ than Fert- cows (17.9 vs. 16.8mm, respectively); however, circulating concentrations of estradiol were not different between genotypes. A greater proportion of Fert- cows ovulated to a silent heat than Fert+ cows (22 vs. 2%, respectively). Of cows that showed behavioral estrus, Fert+ cows had 41% greater mean activity count; however, no difference was seen in mounting behavior between genotypes. These results demonstrate, for the first time, that genetic merit for fertility has pronounced effects on corpus luteum development, progesterone concentration, preovulatory follicle diameter, and behavioral estrus.     

Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.     

Localization and temporal regulation of tissue inhibitor of metalloproteinases-4 in mouse ovary

Tissue inhibitors of metalloproteinases (TIMPs) are potential regulators of tissue remodeling in the ovary. The aim of the present study was to examine the localization and temporal regulation of TIMP-4 protein in the mouse ovary. An induced superovulation model (eCG/hCG) was employed in immature mice to evaluate TIMP-4 protein expression profiles in ovaries collected during the follicular phase, the pre ovulatory period, and the luteal lifespan. Immunofluorescence results indicated that TIMP-4 protein was localized to theca of both antral and preovulatory follicles and adjacent ovarian stroma. After the initiation of luteinization with hCG, TIMP-4 was observed within the luteinizing granulosa cells and persisted throughout the lifespan of the corpus luteum. In the cycling ovary, TIMP-4 signaling localized to corpus luteum from previous estrous cycles, the theca of preovulatory follicles, and appeared to be lower in newly forming corpus luteum. Western analysis further showed that the levels of TIMP-4 increased significantly during the luteinization process of granulosa cells, but no significant change was found among all corpus luteum stages. A putative regulatory mechanism of TIMP-4 expression was identified utilizing an in vitro model. Treatment of cultured granulosa cells with hCG significantly augmented TIMP-4 protein expression levels. Together our data indicate that the luteinization process of granulosa cells is associated with up-regulation of TIMP-4 and that TIMP-4 might play an essential role in maintenance of the luteal function during the whole lifespan of corpus luteum.     

Gonadotrophin surge-induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine periovulatory follicular and luteal tissue

The serine proteinases, tissue-type (tPA) and urokinase (uPA) plasminogen activator, are implicated in the ovulatory processes via their ability to convert plasminogen to its active form, plasmin. One mechanism for regulation of plasmin-directed ovarian extracellular matrix remodelling during follicle rupture and corpus luteum formation is through inhibition of plasminogen activation by the plasminogen activator inhibitors (PAI-1 and PAI-2). The effect of the preovulatory gonadotrophin surge on the temporal and spatial regulation of expression of PAI-1 and PAI-2 mRNA and PAI activity in preovulatory bovine follicles and new corpora lutea collected at 0, 6, 12, 18, 24 and 48 h after a GnRH-induced gonadotrophin surge was examined. Both PAI-1 and PAI-2 mRNAs were upregulated markedly after the gonadotrophin surge, with the highest expression observed in follicles collected at about the time of ovulation (24 h) and in corpora lutea (48 h). PAI-1 mRNA was localized primarily to the thecal layer of preovulatory follicles. In contrast, PAI-2 mRNA was localized specifically to the granulosa cell layer. Significant PAI activity was detected in follicle extracts, but temporal or spatial differences in PAI activity were not detected in response to the gonadotrophin surge. These results indicate that PAI-1 and PAI-2 mRNAs are upregulated in preovulatory bovine follicles after the gonadotrophin surge in a cell-specific way. Regulation of PAI-1 and PAI-2 may help to control plasminogen activator activity associated with ovulation and early corpus luteum formation.     

Effects of synchronization treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid hormone concentrations in lactating dairy cows

Lactating dairy cows (n=57) ≥45 d postpartum at first service were enrolled in a randomized complete block design study to evaluate treatments to synchronize estrus and ovulation. At 10 d before artificial insemination (AI), animals were randomly assigned to 1 of 3 treatments: (1) d -10 GnRH (GnRH1; 10 μg of buserelin, i.m.) and controlled internal drug release insert [CIDR, 1.38 g of progesterone (P4)]; d -3 PGF(2α) (PGF; 25 mg of dinoprost, i.m.); d -2 CIDR out; and AI at observed estrus (CIDR_OBS); (2) same as CIDR_OBS, but GnRH (GnRH2) 36 h after CIDR out and timed AI (TAI) 18 h later (CIDR_TAI); or (3) same as CIDR_TAI, but no CIDR (Ovsynch). Transrectal ultrasound was used to assess follicle size before ovulation and on d 4, 8, and 15 after the presumptive day of estrus (d 0) to measure the corpus luteum (CL). Blood samples were collected to determine concentrations of estradiol (E2; d -10, -9, -3, -2, -1, and 0) and P4 (d -10, -9, -2, -1, 0, 1, 4, 6, 8, 11, and 15). No treatment differences were observed in either circulating concentrations of P4 or the ovulatory response to GnRH1 at the onset of synchronization treatments. Circulating concentrations of P4 were greater for CIDR_OBS and CIDR_TAI compared with Ovsynch at 24 h after CIDR insertion (5.34 and 4.98 vs. 1.75 ng/mL) and immediately before CIDR removal (1.65 and 1.48 vs. 0.40 ng/mL). Peak circulating concentrations of E2 were greater for CIDR_OBS compared with Ovsynch (3.85 vs. 2.39 pg/mL), but CIDR_TAI (2.82 pg/mL) did not differ from either CIDR_OBS or Ovsynch. The interval from PGF injection to peak circulating E2 did not differ between CIDR_TAI and Ovsynch (52.1 vs. 49.8 h). Both CIDR_TAI and Ovsynch, however, had shorter intervals from PGF injection to peak circulating E2 concentrations compared with CIDR_OBS (67.8 h). The diameter of the dominant follicle before ovulation was greater for CIDR_OBS compared with Ovsynch (18.5 vs. 16.0 mm) but CIDR_TAI (17.1 mm) did not differ from either of the other treatments. The mean interval from PGF to ovulation was longer for CIDR_OBS (100.0 h) compared with CIDR_TAI and Ovsynch (84.4 and 83.2 h, respectively). Use of CIDR_OBS resulted in increased preovulatory follicle size and greater circulating concentrations of E2 due to a longer period of preovulatory follicle growth. Progesterone supplementation during synchronization and GnRH on the day before TAI affected ovulatory follicle size, and periovulatory circulating concentrations of P4 and E2. No differences, however, in postovulatory P4 or luteal volume profiles were observed.     

The FecB (Booroola) gene acts at the ovary: in vivo evidence

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.     

Contraception by induction of luteinized unruptured follicles with short-acting low molecular weight FSH receptor agonists in female animal models

During recent decades minor innovative drugs have been developed for the female contraceptive market and they all contain steroidal progestagens (and estrogens) that act centrally and have side effects that can be attributed to this central action. In this study, we present an innovative tissue-specific approach for female contraception by low molecular weight (LMW) FSH receptor (FSHR) agonists, which interact with the FSHR that is dominantly expressed in the granulosa cells. The oral administration of LMW FSHR agonists with a short circulation time, induced formation of luteinized unruptured follicles (LUFs) from the Graafian follicles, thereby preventing the release of the oocyte. The short-acting LMW FSHR compounds were fully agonistic to FSHR (EC(50)=4-5 nM). In an isolated mouse follicle culture, a short incubation period (2 h) resulted in inhibition of follicular rupture, where continuous incubation induced follicle growth. Pharmacokinetics after oral administration showed a surge-like exposure in rats and monkeys. Oral administration of short-acting LMW FSHR agonists inhibited ovulation at 10 mg/kg in rats and guinea pigs by generating LUFs without affecting cyclicity. Also, inhibition of follicular rupture was shown to be reversible within one cycle. Finally, LUFs were induced without affecting the hormonal cyclicity in cynomolgus monkeys, a mono-ovulatory species. In healthy women LUF formation occurs naturally, with a LUF acting as corpus luteum that produces enough progesterone to ensure normal menstrual cyclicity. Together with the presented data this indicates that the innovative approach with short-acting LMW FSHR agonists could lead to oral contraception for females at the ovarian level.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Recent advances in corpus luteum physiology

Development, maintenance, and regression of the corpus luteum have been investigated for many years. However, endocrine and cellular mechanisms regulating progesterone synthesis and secretion remain unclear. Because comprehensive reviews of factors affecting luteal function have been published recently, this paper discusses several emerging concepts that may be important in understanding the regulation of luteal progesterone synthesis and secretion. Concepts discussed include preovulatory follicular determinants of subsequent luteal function, hormonal stimulation of progesterone synthesis, effect of different luteal cell types on progesterone secretion, and role of secretory granules in luteal function.     

Prostaglandin F2α and gonadotropin-releasing hormone administration improve progesterone status, luteal number, and proportion of ovular and anovular dairy cows with corpora lutea before a timed artificial insemination program

The objective of this research was to increase the proportion of cows with at least 1 functional corpus luteum (CL) and elevated progesterone at the onset of the timed artificial insemination (TAI) protocol. Postpartum Holstein cows in one herd were stratified by lactation number at calving (September 2009 through August 2010) and assigned randomly to 2 treatments: 1) Presynch-10 (n=105): two 25-mg injections of PGF(2α) (PG) 14 d apart (Presynch); and 2) PG-3-G (n=105): one 25-mg injection of PG 3 d before 100-μg GnRH (Pre-GnRH) injection, with the PG injection administered at the same time as the second PG injection in the Presynch-10 treatment. Cows were enrolled in a TAI protocol [Ovsynch; injection of GnRH 7 d before (GnRH-1) and 56 h after (GnRH-2) PG injection with AI 16 to 18 h after GnRH-2] 10 d after the second or only PG injection. Blood samples for progesterone or estradiol analyses were collected on median days in milk (DIM): 36, 39, 50, 53 (Pre-GnRH), 60 (GnRH-1), 67 (PG), 69 (GnRH-2), and 70 (TAI). Ovarian structures were measured by ultrasonography on median DIM 53, 60, 67, 69, and 6 d post-TAI to determine follicle diameters, ovulation response to GnRH, or both. Although progesterone concentration did not differ between treatments before Pre-GnRH injection, the proportion of cows with at least 1 CL tended to be greater for PG-3-G than Presynch-10 cows, and more PG-3-G cows ovulated after Pre-GnRH injection than ovulated spontaneously in Presynch-10. Further, the diameter of follicles that ovulated tended to be smaller in PG-3-G than in Presynch-10 cows after Pre-GnRH injection. At GnRH-1, the proportion of cows with progesterone ≥1 ng/mL, the number of CL per cow, and the proportion of cows with at least 1 CL were greater for PG-3-G than Presynch-10. Neither follicle diameter nor percentage of cows ovulating after GnRH-1 differed between treatments. At PG injection during the week of TAI, progesterone concentration and the proportion of cows with progesterone ≥1 ng/mL tended to be greater for PG-3-G than Presynch-10, and PG-3-G had more CL per cow than Presynch-10. No ovarian characteristics differed between treatments after GnRH-2, including progesterone concentration, number of CL per cow, and total luteal volume 7 d after GnRH-2. Many of the previous ovarian traits were improved in both ovular and anovular cows after PG-3-G compared with Presynch-10. Pregnancies per AI at d 32 and 60 were only numerically greater for PG-3-G than for Presynch-10 cows, largely because of differences detected during months without heat stress. We concluded that the PG-3-G treatment increased ovulation rate and luteal function 7 d before the onset of Ovsynch, resulting in improved follicular synchrony and predisposing potentially greater pregnancies per AI in lactating dairy cows.     

Changes in fibroblast growth factor 2 and its receptors in bovine follicles before and after GnRH application and after ovulation

The aim of this study was to evaluate the expression pattern of fibroblast growth factor 2 (FGF2), its receptor variants (FGFR1IIIc, FGFR2IIIc) and nucleolin in time-defined follicle classes before and after GnRH application and after ovulation in the cow. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 4, 10, 20 and 25 h (follicles) and 60 h (new CL) relative to injection of GnRH to induce an LH surge (n = 5 animals per group). The expressions of FGF2 and FGFR1IIIc mRNA were significantly up-regulated only in the follicle group 4 h after GnRH (during the LH surge) with a significant down-regulation immediately afterwards. Western blot analyses showed two protein bands with at 22 and 18 kDa with apparent up-regulation beginning with the LH surge (4 h) and maximum levels 20 h after GnRH. FGF2 protein in follicles collected at 0 h (before LH surge) was localised in theca tissue (endothelial and pericytes of blood vessels) but not in granulosa cells (GCs). The FGF2 staining (by immunohistochemistry) pattern changed dramatically after the LH surge for a short period (about 2 days) and FGF2 protein was localised dominantly in the nucleus of many GCs, while most capillary endothelial cells were FGF2 immunonegative. In conclusion, the novel observation of FGF2 up-regulation and the distinct change in FGF2 localisation from theca (cytoplasm of endothelial cells) to the nucleus of GCs after the LH surge may be important for survival of GCs or for the transition of the GCs to luteal cells.     

Graduate Student Literature Review: Effects of human chorionic gonadotropin on follicular and luteal dynamics and fertility in cattle

Circulating progesterone concentrations during the growth of the ovulatory follicle and early embryo development have been positively associated with embryo quality and survival and pregnancy success. As a potent luteotropic agent with LH-like activity, human chorionic gonadotropin (hCG) has been tested in different studies to improve pregnancy outcomes by increasing circulating progesterone concentrations during the growth of the ovulatory follicle or early embryonic development. Nevertheless, hCG has produced inconsistent, contradictory, and intriguing results. Furthermore, recent research indicates that hCG, when used before artificial insemination, may affect physiological events necessary for the ovulation of a viable oocyte. In addition, the use of hCG-inducing accessory corpus luteum during the estrous cycle seems to disturb luteolysis and follicle and luteal dynamics during the estrous cycle. This literature review discusses past and current research exploring the effects of hCG on the estrous cycle characteristics and pregnancy per artificial insemination and embryo transfer.     

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation,follicle dynamics,and circulating follicle-stimulating hormone in lactating dairy cows

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 μg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (～18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5–11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.     

Ovarian function in ewes after treatment with mifepristone early during the oestrous cycle

The aim of this study was to determine whether endogenous progesterone regulates synthesis and secretion of luteal oxytocin. In Expt 1, mature ewes (n = 5 per group) were assigned randomly to control or mifepristone (RU486) treatment groups. Ewes were injected s.c. twice a day with vehicle or 10 mg RU486 on days 5-7 of the oestrous cycle (oestrus = day 0). On day 8, after an i.v. injection with prostaglandin F(2alpha) (250 microg cloprostenol), venous blood samples were collected at frequent intervals to determine plasma oxytocin concentrations. Plasma oxytocin concentrations of RU486-treated ewes were not significantly different from those of control ewes. In Expt 2, ewes were injected s.c. each day with vehicle or 175 mg RU486 on days 2-5 of the oestrous cycle followed by administration of prostaglandin F(2alpha) on day 6. Four of five RU486-treated ewes showed 'split-oestrus' (oestrous behaviour for 36 h and then again at 84-108 h after the onset of initial oestrus). There was no significant difference in mean plasma oxytocin or progesterone concentrations between treatment groups. The mean masses of mature corpora lutea from control and RU486-treated ewes on day 6 of the oestrous cycle did not differ significantly (394.8 +/- 28.8 versus 319.5 +/- 48.3 mg). RU486-treated ewes contained mature corpora lutea, new corpora lutea (two of four ewes) and preovulatory follicles (>or= 10 mm, two of four ewes). The average interoestrous interval for RU486-treated ewes was 9 days more than that for control animals (26.2 +/- 2.9 versus 17 +/- 0.5 days; P < 0.025).     

Effect of intrafollicular indomethacin injection on gonadotropin surge-induced expression of select extracellular matrix degrading enzymes and their inhibitors in bovine preovulatory follicles

A growing body of evidence supports an obligatory role for intrafollicular prostanoids in the mechanism of ovulation. However, the prostanoid-dependent mediators of the follicular extracellular matrix degradation required for ovulation are unknown. The objectives of this study were to determine the cellular compartment(s) in which the gonadotropin surge-induced regulation of select extracellular matrix degrading enzymes and their cognate inhibitors occurs in bovine preovulatory follicles, and to test whether such regulation is blocked by intrafollicular administration of the prostanoid synthesis and ovulation inhibitor, indomethacin (INDO). Follicular fluid prostaglandin E2 concentrations were elevated in diluent-treated follicles before ovulation (24 h after GnRH injection), but the increase was blocked in INDO-treated follicles. Real-time PCR analysis revealed the specific follicular cell types where gonadotropin surge-induced increases in mRNA abundance for members of the matrix metalloproteinase/tissue inhibitor of metalloproteinase and plasminogen activator families occurred. INDO treatment increased thecal cell mRNA for tissue inhibitor of metalloproteinase-4 and its protein abundance in the apex of preovulatory follicles before ovulation, but suppressed granulosal cell mRNA and activity for tissue plasminogen activator in follicular fluid and the follicle apex. Plasmin activity was also suppressed in the follicular fluid of INDO-treated follicles. Effects of INDO injection on select matrix metalloproteinases were not observed. The results suggest that gonadotropin surge-induced regulation of tissue inhibitor of metalloproteinase-4 and tissue plasminogen activator may be prostanoid dependent, and support a potential role for increased tissue plasminogen activator expression and decreased tissue inhibitor of metalloproteinase-4 expression in the mechanism of ovulation.     

E- and N-cadherin expression and distribution during luteinization in the rat ovary

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.     

Effects of whole cottonseed diet and recombinant bovine somatotropin on ovarian follicles in lactating dairy cows

The effects of whole cottonseed (WCS) in the diet and the administration of bovine somatotropin (bST) on ovarian follicular dynamics and plasma progesterone (P4) concentrations were examined in cows during a period of synchronized follicular growth. Lactating Holstein cows (n = 28) were randomly assigned to treatments in a 2 x 2 factorial arrangement. Diets consisted of WCS (15% of dry matter) or no WCS, and bST at a dose of 0 or 208 mg/14 d. Dietary treatments began within 24 h of calving and bST treatments began within 7 d postpartum. Cows received GnRH at 65 +/- 3 d postpartum (d 0), PGF2alpha, (d 7), a second dose of GnRH (d 9), and were inseminated 16 h later (d 10). Ovarian changes were monitored daily by ultrasonography from d 0 to 9. On d 9,93% of cows had a preovulatory follicle and 86% ovulated. For Class 2 (6 to 9 mm) follicles, a diet x bST interaction was detected, with bST stimulating Class 2 follicles in cows fed WCS, but not in cows on the control diet. Neither diet nor bST affected numbers of Class 1 (2 to 5 mm) or Class 3 (> or = 10 mm) follicles or sizes of the subordinate and dominant follicles. During the luteal phase of the cycle, lactating cows fed WCS tended to have elevated concentrations of plasma P4, whereas bST was without effect. Plasma concentrations of high-density lipoprotein cholesterol were increased in cows fed WCS. Number and diameter of corpora lutea did not differ among treatments.