Plasma progesterone levels in sows with induced cystic ovarian follicles.

Large multiple cystic ovarian follicles were induced in three sows and small multiple cystic ovarian follicles were induced in three other sows by injections of adrenocorticotrophic hormone (ACTH) during the follicular phase of the oestrous cycle. Plasma progesterone levels in sows with large cysts were relatively high, while those of sows with small cystic follicles were low. Removal of the ovaries with large cysts from one sow resulted in a precipitous drop in progesterone levels indicating that the large cysts were primarily the source of progesterone. The method by which ACTH may induced cystic ovaries was also investigated. Plasma progesterone levels in two ovariectomised sows during ACTH treatment fluctuated markedly during a 24 h period achieving peak values of 4 or 5 ng/ml. The results suggest that progesterone of adrenal cortical origin may be a factor in the development of the cystic ovarian condition.     

Estrogen receptors in the uterus and ovarian follicles of gilts treated with dihydrotestosterone

Experiments were conducted to evaluate expression of the estrogen receptor (ER) alpha and ERbeta genes in the uterus and ovarian follicles of gilts treated with 5alpha-dihydrotestosterone (DHT) during the follicular phase of the estrous cycle. This DHT treatment has enhanced ovulation rate but decreased blastocyst survival in previous experiments. Gilts received daily i.m. injections of 10 mg of DHT from day 13 (day 0 = onset of estrus) to day 18 (experiment 1), or from day 13 to 16 (experiment 2) of the estrous cycle. Gilts that served as controls received vehicle. The ovaries and a portion of uterine horn were surgically removed 24 h after the last treatment. Administration of DHT from day 13 to 18 of the estrous cycle decreased uterine wet weight (tendency, P = 0.10), and the relative amounts (ratios to ribosomal protein L19) of endometrial mRNA for the estrogen-responsive gene complement component C3. Gilts receiving DHT had greater amounts of ERbeta mRNA in the endometrium than those treated with vehicle in both experiments, but DHT did not alter the overall amounts of endometrial ERalpha mRNA. Immunohistochemical (IHC) analysis demonstrated that DHT did not alter the relative amounts of ERalpha in the myometrium, glandular and luminal epithelia and endometrial subepithelial stroma. In the ovary, amounts of ERalpha and ERbeta mRNAs in surface walls of follicles > or =6 mm in diameter were not altered by DHT treatments, however, DHT treatment from day 13 to 16 decreased the amounts of immunoreactive ERalpha in the theca interna at the surface walls of day 17 follicles (experiment 2). The amounts of immunoreactive ERalpha were greater in the granulosa than in the theca interna, and within cell type, the amounts of ERalpha were greater at the surface than at the basal region of the follicles, with the exception of the theca interna in follicles evaluated on day 19 (experiment 1). Treatment of gilts with DHT during the follicular phase of the estrous cycle increased ERbeta mRNA in the endometrium and influenced the amounts of immunoreactive ERalpha in ovarian follicles in a cell type-, day of development- and region-specific manner.     

Induction of ovarian cystic follicles in sheep

Cystic follicles are a significant cause of infertility in women, dairy cattle and sheep. Sheep were used as a model to identify factors that may elicit formation of cystic follicles. Insulin resistance and elevated LH activity were tested in overweight ewes because of associations among these factors and the formation of cystic follicles. Sheep were synchronized using a progesterone-releasing pessary and insulin resistance was induced during the synchronization period through administration of bovine somatotropin. Following removal of pessaries follicular growth was stimulated by treatment with eCG or eCG and hCG (PG-600). Follicular growth was monitored via daily transrectal ultrasonography and blood samples were collected for hormonal analyses. Six of 18 ewes had a subnormal or absent preovulatory gonadotropin surge and developed cystic follicles. Neither insulin resistance nor elevated LH activity were associated with formation of cystic follicles. Ewes that developed cystic follicles were heavier (93 +/- 4 kg) than ewes that ovulated (81 +/- 3 kg; P = 0.02). Furthermore, following pessary removal and initiation of daily ultrasonography, ewes that developed cystic follicles lost body weight (-3 +/- 1%), while ovulatory ewes continued to gain body weight (1 +/- 1%; P = 0.005). It is speculated that in heavy ewes metabolic factors associated with acute body weight loss inhibit the positive feedback of estradiol and thereby suppress the preovulatory gonadotropin surge leading to formation of cystic follicles.     

Colour Doppler sonography of cystic ovarian follicles in cows

The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B-mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue. Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.     

Efficacy of intravaginal progesterone administration combined with prostaglandin f(2alpha) for cystic ovarian disease in Japanese Black cows

The objective of this study was to investigate cystic ovarian disease (COD) in commercial Japanese Black cows and to evaluate the efficacy of 7-day insertion of an intravaginal progesterone insert (CIDR) combined with prostaglandin F(2alpha) (PGF(2alpha)) at CIDR removal. Experiment 1 was conducted to group cysts into 4 patterns based on alteration of plasma progesterone (P(4)) concentrations on d -7 and d 0 (=CIDR insertion) with 1.0 ng/ml as the cut-off level by ultrasonographic examination of 28 cows with COD that were >or=40 days postpartum and anoestrous after calving. In Experiment 2, a total of 55 cows under the same conditions as in Experiment 1 were utilized, and the same regimen as in Experiment 1 was performed without 7 days of pre-observation before treatment. As a result, 92.9% of CLs on d 21 were highly formed in Experiment 1 and 83.6% were highly formed in Experiment 2. The conception rates within 60 days after CIDR removal were also satisfactory high and were 71.4% and 54.5%, respectively. There were no differences in any overall reproductive parameters between Experiments 1 and 2 (P>0.05). The average days between CIDR removal and conception were 24.4 +/- 5.3 and 24.0 +/- 6.5 days, respectively (P>0.05); therefore, the conception dates of the cows in Experiment 2 were at least 7 days earlier compared with Experiment 1. In conclusion, treatment with a CIDR and PGF(2alpha) against COD could minimize the risk of incorrect treatment and provide sufficient reproductive performance in Japanese Black cows.     

Comparison of cadherin and integrin localization in bovine cystic and healthy follicles

As stage progresses in the cystic follicle, granulosa cells are lost. We hypothesized that the granulosa and theca interna layers are detached in association with weakened expression of cell adhesion molecules such as cadherin (cell–cell adhesion) and integrin (cell–extracellular matrix adhesion) in cystic follicles. To elucidate this hypothesis, we immunolocalized these molecules in the granulosa and theca interna and compared them between cystic and small healthy follicles. Sections were immunostained with cadherin and integrin β₁ antibodies and their localizations were compared. Cadherin‐positive reaction was seen in the cytoplasma of all granulosa cells. No increase in the frequency of cadherin‐positive area in the granulosa layers and the intensity of cadherin immunoreaction in the theca interna was detected in cystic follicles compared with healthy ones. A dense immunoreaction product of integrin β₁ was detected in the theca interna in both cystic and healthy follicles. Intensity of integrin β₁‐immuno reaction in the granulosa layers and integrin β₁‐positive area in the theca interna was significantly lower in the cystic follicle than in the healthy follicles. These results suggest that granulosa and theca interna cells are detached while maintaining the cell–cell adhesion, resulting in the consequent loss of these layers from the cystic follicle.     

Ovarian and endocrine responses associated with the treatment of cystic ovarian follicles in dairy cows with gonadotropin releasing hormone and prostaglandin F2alpha, with or without exogenous progesterone

The objectives of this observational study were to document ovarian and endocrine responses associated with the treatment of cystic ovarian follicles (COFs) in dairy cows, using gonadotropin releasing hormone (GnRH) and prostaglandin F2alpha (PGF) with or without exogenous progesterone. A secondary objective was to determine pregnancy establishment following synchronization of ovulation and timed insemination in cows diagnosed with COFs. In trial I, 18 Holstein cows diagnosed with COFs received 2 injections of 100 microg GnRH, 9 d apart, with 25 mg PGF given 7 d after the 1st GnRH. A new follicle developed in all 18 cows after the 1st GnRH, and 83% of cows ovulated following the 2nd GnRH. Cows were inseminated 16 h after the 2nd GnRH. Of the 17 cows available for pregnancy diagnosis, 7 were confirmed pregnant. In trial II, 8 cows with COFs received GnRH and an intravaginal progesterone device (CIDR) concurrently, then PGF 7 d later. The CIDR was removed 2 d after PGF administration. Plasma estradiol concentrations declined following CIDR insertion. In all cows, a new follicle developed following GnRH treatment; estradiol-surge and estrus occurred spontaneously after CIDR-removal. Seven of 8 cows ovulated the new follicle. In dairy cows diagnosed with COFs, treatment with GnRH followed by PGF 7 d later, with or without exogenous progesterone, resulted in the recruitment of a healthy new follicle; synchronization of ovulation and timed insemination resulted in a 41% pregnancy rate.     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Role for LDL in estradiol-synthesis capacity of bovine ovarian follicles

The steroidogenic capacity of several tissues has been shown to be regulated by lipoproteins. However, the ability of the various lipoprotein classes to affect steroid production in general and estradiol-synthesis in particular has not been established in bovine ovarian cells. In previous studies, we described alterations in the lipoprotein profiles in follicles of different sizes and corresponding changes in lipoprotein-receptor gene expression. In the present study, the effect of low-density lipoprotein (LDL)-enriched medium on aromatization competence of whole ovarian follicles was determined in vitro. Gene expression of aromatase increased and that of selective-uptake receptors decreased in the presence of LDL. These results suggest a role for LDL availability in folliculogenesis.     

Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.     

Comparison of surface diameters and dissected diameters of bovine ovarian follicles

Comparisons were made between diameters of 54 bovine follicles greater than 5.9 mm from 32 pairs of ovaries measured on the ovarian surface and diameters of the same follicles subsequently dissected from the ovaries. Seventy-eight percent of follicles measured on the ovarian surface were within 1.9 mm of the size measured after dissection. The remaining 22% of follicles measured on the surface had diameters recorded that were 2.0 to 3.9 mm different than their diameter after dissection. Surface diameter tended to underestimate dissected diameter for small follicles (less than 8.0 mm) and to overestimate dissected diameter for large (greater than or equal to 12.0 mm) follicles. The correlation coefficient between surface and dissected follicular diameters was .83. We conclude that measuring the diameter of the largest follicles on the ovarian surface and after dissection yield approximately equivalent results.     

Tumor necrosis factor-alpha (TNF alpha) inhibits progesterone and estradiol-17beta production from cultured granulosa cells: presence of TNFalpha receptors in bovine granulosa and theca cells

The aim of this study was to investigate whether functional tumor necrosis factor-alpha (TNFalpha) receptors are present in the granulosa cells and the cells of theca interna (theca cells), obtained from bovine follicles classified into one of three groups. Each group was defined as either small vesicular ovarian follicles (small follicles; 3-5 mm in diameter), preovulatory mature ovarian follicles (preovulatory follicles) or atretic follicles (12-18 mm) according to gross examination of the corpus luteum in the epsilateral or contralateral ovary and the uterus (size, color, consistency and mucus), and the ratio of progesterone (P(4)) and estradiol-17beta (E(2)) concentrations in follicular fluid. A Scatchard analysis showed the presence of a high-affinity binding site on both granulosa and theca cells from all follicles examined (dissociation constant: 4.7 +/- 0.15 to 6.9 +/- 1.40 nM). Moreover, TNFalpha receptor concentrations in granulosa and theca cells obtained from atretic follicles were significantly higher than those in the cells from preovulatory follicles (P<0.05). Exposure of cultured granulosa cells from small antral follicles to recombinant human TNFalpha (rhTNFalpha; 0.06-6 nM) inhibited E(2) secretion in a dose-dependent fashion (P<0.01), but did not affect P(4) secretion. In addition, rhTNFalpha inhibited follicle stimulating hormone-, forskolin- or dibutylyl cyclic AMP-induced P(4) and E(2) secretion by the cells (P<0.01). These results indicate the presence of functional TNFalpha receptors in bovine granulosa and theca cells in small, preovulatory and atretic follicles, and suggest that TNFalpha plays a role in regulating their secretory function.