Localization of Estrogen Receptor Beta (ERß) mRNA within Different Bovine Ovarian Follicles

The oestrogen receptor beta (ERß) is largely distributed in the ovary of many species but data for the bovine ovary are scare. Therefore, the expression of ERß mRNA in the different follicles of the bovine ovary was studied using in situ hybridization. Ovarian tissue sections of three cows with different plasma progesterone concentrations were used (cow 1: 3.50 ng/ml; cow 2: 1.00 ng/ml, cow 3: 0.35 ng/ml). A 602 bp fragment of ERß mRNA was cloned, sequenced and digoxigenin (DIG)‐labelled. Subsequently, in situ hybridization was performed by incubating the sections with the DIG‐labelled RNA anti‐sense probe. For the semi‐quantitative evaluation of ERß mRNA expression the ERß mRNA score (SER) was determined for the different follicular cell types using the formula: SER = 0.n0 + 1.n1+ 2.n2 + 3.n3 with n0, n1, n2, n3 indicating the percentage of cells exhibiting a staining intensity 0 (absent), 1 (weak), 2 (moderate) or 3 (strong), respectively. High ER mRNA levels were noticed in primordial and primary follicle cells, and suggest a role of ER mRNA in early folliculogenesis. A lower SER was observed in the granulosa cells of secondary and tertiary follicles. This significant difference in the SER of follicle cells during follicular growth may be associated with cell proliferation. In obliterative and cystic atretic follicles high SER were observed, although ERß mRNA levels in obliterative follicles showed much inter‐individual variation. This is suggestive for ERß mediated oestrogen action in atretic follicles. In the corpora lutea moderate ERß mRNA levels were noticed. Our findings are in accordance with studies in the ewe in which corpora lutea cells synthesize estrogen. These preliminary findings will be further elaborated in a higher number of cows to examine the role of ERß in the ovary throughout the oestrus cycle.     

Cell-specific localization of oestrogen receptor beta (ESR2) mRNA within various bovine ovarian cell types using in situ hybridization

The localization of oestrogen receptor beta (ESR2) mRNA, in this article denominated as (ERbeta) mRNA, was examined using in situ hybridization in the ovaries of randomly selected cows, irrespective of the cycle stage of the animals. A 602-bp fragment of ERbeta mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Semi-quantitative evaluation showed that the scores for ERbeta mRNA were moderate to high in the follicle cells of both primordial and primary follicles, but lower in granulosa cells of secondary follicles. In vital tertiary follicles, the total ERbeta mRNA expression was low but varied between the different animals. In both obliterative and cystic atretic follicles, high to moderate ERbeta mRNA scores were noticed in the granulosa cells. The stroma cells surrounding primordial and primary follicles and the theca cells of secondary follicles showed moderate ERbeta mRNA levels, whereas the ERbeta mRNA score in theca interna and theca externa cells of vital tertiary follicles was distinctly higher. In the theca cells of atretic follicles the score was even higher. Cells of corpora hemorrhagica and corpora lutea had moderate ERbeta mRNA scores, while higher scores were seen in cells of corpora albicantia. Cells of the surface epithelium had a moderate score for ERbeta mRNA, whereas cells of the tunica albuginea and deep stroma showed high ERbeta mRNA scores. The present findings have clearly established a cell-specific localization of ERbeta mRNA in several cell types in the bovine ovary.     

Localization of oestrogen receptors within various bovine ovarian cell types at different stages of the oestrous cycle

In the present study oestrogen receptor alpha(ERalpha) and oestrogen receptor beta (ERbeta) mRNA were localized in various ovarian cell types of 23 cows at different stages of the oestrous cycle. ERalpha was detected by immunohistochemistry and the localization of ERbeta mRNA was examined using in situ hybridization. The immunostaining of ERalpha was low in the ovarian follicles, tunica albuginea and surface epithelium, but high in cells of the deep stroma and superficial stroma, which indicates a functional role of ERalpha in the cells surrounding the follicles. In contrast, ERbeta mRNA scores were low to moderate in primordial and primary follicles, and increased with the development of the follicle. ERbeta mRNA scores were higher in cystic follicles than in obliterative follicles. In the corpora lutea and corpora albicantia the scores for ERbeta mRNA were moderate. Furthermore, in the corpora lutea, ERbeta mRNA levels showed cyclic variations and were low during early dioestrus. The correlation between plasma progesterone levels and the score for ER was low and negative in all ovarian cell types. This study demonstrates the predominant role of ERbeta over ERalpha in bovine ovarian structures. Furthermore, the colocalization of both ERbeta mRNA and ERalpha in most cell types suggests possible interactions between both ER subtypes.     

Erratum

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non‐pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down‐regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.     

The Immunohistochemical Localization of Desmin and Smooth Muscle Actin in the Ovary of the African Giant Rat (Cricetomys gambianus) During the Oestrous Cycle

The aim of this study was to describe the distribution of smooth muscle actin and desmin immunopositive cells in the ovary of the giant rat. In addition, the study describes the morphological changes in the ovary of this species during the oestrous cycle. Healthy secondary and tertiary follicles dominated the ovary during pro-oestrus and oestrus. The theca externa of the tertiary follicles was immunopositive for smooth muscle actin, but immunonegative for desmin. Oestrus was also characterized by the presence of corpora haemorrhagica, which had an outer layer of smooth muscle actin immunopositive cells. Differentiating corpora lutea were observed during metoestrus. A further notable feature of the ovary during metoestrus was the presence of numerous atretic secondary and tertiary follicles. In the later stages of atresia, the follicles were infiltrated by desmin and smooth muscle actin immunopositive cells. Dioestrus was characterized by the presence of non-regressing and regressing corpora lutea. Immunostaining for smooth muscle actin was demonstrated in the enclosing layer of the corpora lutea, as well as in the tunica media of blood vessels within the corpora lutea. The results of this study have shown that morphological changes in the ovary of the giant rat during the oestrus cycle are similar to those of laboratory rodents. Furthermore, the results of the immunohistochemical study indicate that the perifollicular distribution of desmin and smooth muscle actin cells changes during follicular development and atresia.     

Effects of Maternal Undernutrition on the Hypothalamic–Pituitary–Gonadal Axis Function in Female Sheep Offspring

A study was conducted to evaluate the effects of maternal undernutrition on the hypothalamic–pituitary–gonadal axis in female sheep offspring. Pregnant ewes were fed to 100% throughout pregnancy (Control) or to 50% from 0 to 30 (R1) or from 31 to 100 days of gestation (R2). Female lambs were selected and fed to appetite throughout the study. At 2, 5.5 and 10 months of age a GnRH challenge was conducted. At the age of 10 months lambs were synchronized and blood samples were collected at 3 h intervals for 72 h following sponge removal. At slaughter (10 months) ovaries were removed and examined macroscopically. Maternal undernutrition did not affect the time of the onset of puberty, defined as the first increase in plasma progesterone concentrations ≥1 ng/ml. The magnitude of the pre-ovulatory gonadotrophin surge and the time to surge were unaffected by treatment. The LH and FSH response to GnRH challenge did not differ between groups at 2 and 5.5 months but at 10 months of age a higher (p < 0.05) FSH response was found in R1 group. Although the total number of visible follicles and corpora lutea did not differ between groups, a significant higher (p < 0.05) number of small (2–3 mm diameter) follicles in R1 group and a significant lower number (p < 0.05) of corpora lutea with diameter 8–11 mm and not even one with diameter >12 mm were detected in the ovaries of R2 lambs. In conclusion, maternal undernutrition during the first month of pregnancy resulted in increased pituitary sensitivity to GnRH and increased number of small follicles in the ovary, while during mid to late gestation resulted in a reduction of large corpora lutea in female offspring.     

Non Cat-Like Ovarian Cycle in the Eurasian and the Iberian Lynx – Ultrasonographical and Endocrinological Analysis

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 ± 1.3 ng/ml in Eurasian and 6.1 ± 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.     

Characterization of ovine follicles destined to form subfunctional corpora lutea

A study was done to test whether ovulatory follicles destined to form subfunctional corpora lutea differed from normal ovulatory follicles in steroidogenic function. Twenty-five ewes were treated with prostaglandin F2 alpha on d 11 of the estrous cycle, then unilaterally ovariectomized before (n = 13) or after (n = 12) the surge of luteinizing hormone (LH) at the induced estrus to collect "control" follicles, which would have produced normal corpora lutea. In 15 ewes, the second ovary was removed 63 to 84 h later to collect "treated" follicles before (n = 7) or after (n = 8) the second expected surge of LH. Five ewes (control) were allowed to ovulate from the remaining ovary at first estrus and another five (treated) at the second estrus (3 to 4 d later). Treated ewes had lower serum progesterone than control ewes during the ensuing cycle (P less than .05). Treated follicles contained less estradiol in the theca (4.4 +/- .6 vs 10.0 +/- 2.5 ng; P less than .05), less androstenedione (.1 +/- .1 vs 1.0 +/- .2 ng) and estradiol (.5 +/- .1 vs 2.9 +/- 2.2 ng) in the granulosa (P less than .05) and less progesterone in the follicular fluid (.8 +/- .4 vs 3.3 +/- .8 ng; P less than .05) than control follicles, when removed before the surge of LH. Follicles removed after the surge of LH did not differ. In conclusion, ovulatory follicles with low steroidogenic function became corpora lutea that secreted lower-than-normal quantities of progesterone.     

Cellular Localization of Inhibin alpha-subunit, PKB/Akt and FoxO3a proteins in the ovaries of minipigs

Experiments were conducted to examine the cellular localization of inhibin alpha-subunit, protein kinase B (PKB/Akt), and FoxO3a proteins in the ovaries of minipigs, Chinese Xiang pigs, by immunohistochemistry. The results indicated that inhibin alpha-subunits were localized in the granulosa cells of follicles at all stages but were not localized in corpora lutea. PKB was localized in the granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles, but were not localized in atretic follicles and corpora lutea. FoxO3a was localized in the granulosa cells of follicles at all stages and was extensively localized in the cytoplasma of the luteinized granulosa cells of corpora lutea. Together, the stage- and cell-specific expression patterns of inhibin alpha-subunit, FoxO3a, and PKB suggest that these proteins might play potential roles in follicular development, atresia, and luteinization in the minipig.     

Angiogenesis in The Ovary – The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow – An Overview

In the ovary, the development of new capillaries from pre‐existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin‐like growth factor (IGF), angiopoietin (ANPT) and hypoxia‐inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol‐17‐beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT‐qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF‐A, FGF‐2, IGF‐1 and IGF‐2, ANPT‐2/ANPT‐1 and HIF‐1‐alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.     

Puncture and drainage of the subordinate follicles at timed artificial insemination prevents the risk of twin pregnancy in dairy cows

The objective of this study was to determine whether fluid drainage from the subordinate follicles by ovum pickup procedures prevents the risk of twin pregnancy without reducing the fertility of the cow. Lactating dairy cows with at least two follicles over 12 mm diameter located one on each ovary and selected from synchronized groups for fixed‐time insemination were assigned to a Control (n = 49) or Drainage (n = 49) group. The largest follicle was considered as the dominant follicle. Draining of all subordinate follicles (≥10 mm) was performed in the Drainage group. All drained follicles developed as a corpus luteum 7 days later. In the Control cows, the presence of two corpora lutea (55.1%) was lower (p = .003) than those in the Drainage cows (87.8%), whereas the incidence of twin pregnancies was 50% and 0% for the Control and Drainage groups, respectively (p < .001). Draining did not affect the pregnancy rate. These results indicate that puncture and drainage of the subordinate follicles at insemination may eliminate the risk of twin pregnancies and reduce the risk of subsequent pregnancy loss by increasing the incidence of additional corpora lutea.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

Ovarian function in the cycling cow: relationship between gonadotropin binding to theca and granulosa and steroidogenesis in individual follicles

Thirty-six Angus and Angus crossbred cows were used in a 3 x 2 x 2 factorial experiment designed to determine the effects of stage of the estrous cycle (day 6, 12 or 18), a long term with a single ovary (2 years) and side of previous ovulation or remaining ovary on the development of large ovarian follicles (LF; diameter greater than or equal to 8 mm). Follicular fluid estradiol-17 beta (FFE) concentration, localization of gonadotropin binding sites and granulosa cell condition were determined for each LF by radioimmunoassay, autoradiography and histological examination, respectively. No side differences were noted. However, the sample was biased by the use of equal numbers of left and right side ovulators. Compared to intact controls, one-ovary cows showed 100% compensation of LF development. Eighty-five percent of the LF studied bound follicle stimulating hormone (FSH) in the granulosa and human chorionic gonadotropin (hCG) in the theca. Thirty-eight percent also bound hCG in the granulosa. Both binding patterns were found on all days studied. Atretic follicles had lower (P less than .05) FFE concentrations than healthy follicles (r = -.59). The number of LF per cow increased (P less than .05) from day 6 (1.3) to days 12 (1.8) and 18 (2.1). Conversely, their FFE content per cow decreased (P less than .05) from 109 ng/cow on day 6 to 20 ng on; day 12 and 34 ng on day 18. The data suggested the emergency of two LF classes on day 18, one preovulatory, with exceptionally high FFE levels, the other atretic or becoming atretic, with low FFE levels.     

Endocrine profiles associated with life span of induced corpora lutea in postpartum beef cows

Two experiments were designed to examine whether hormonal profiles were related to luteal life span in pluriparous postpartum anestrous beef cows. Cows (Exp. 1, n = 34; Exp. 2, n = 23) received norgestomet (N) for 9 d or served as controls (C). Each cow received 1,000 IU human chorionic gonadotropin (hCG) 48 h after removal of N (d 0). Blood samples collected every 15 min for 8 h on d -5, 3 and 5 (Exp. 1) or on d -10 and -1 (Exp. 2) were assayed for luteinizing hormone (LH) and follicle stimulating hormone (FSH). Cortisol was determined in hourly samples collected on d -5 and in samples collected every 2 min during suckling on the same day (Exp. 1). Concentrations of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) were determined in samples collected at 15-min intervals for 2 h on d -5, 3, 5 and 10 (Exp. 1). Estradiol-17 beta was measured in samples collected on d -5 (Exp. 1) or on d -10 and -1 (Exp. 2). Life span of induced corpora lutea was longer (P less than .05) in N than C cows. Percentages of N cows in which corpora lutea, formed in response to hCG, exhibited a normal life span were 83% on farm 1 and 25% on farm 2 (Exp. 1), and 90% (Exp. 2), compared with 0% in C cows. Concentrations of FSH were not affected by N but were lower (P less than .05) on d -5 in cows on farm 2 (.6 +/- .1 ng/ml) than in cows on farm 1 (.8 +/- .1 ng/ml). On d -5, a treatment X farm interaction (P less than .05) for mean LH was observed and frequency of pulses of LH was higher (P less than .01) in N than C cows (2.7 +/- .4 vs. .8 +/- .8 pulses/8 h). Neither cortisol nor PGFM was affected by N. Estradiol was increased in d -1 (6.1 +/- .5 vs 2.6 +/- .8 pg/ml; P less than .01) by N. It is suggested that pre-treatment with N enhanced life span of induced corpora lutea, in part, by influencing secretion of LH and development of follicles, but a threshold concentration of FSH was required for N to exert this effect.     

Ovarian morphological conditions and the effect of injection of human chorionic gonadotropin on ovulation rates in prepuberal gilts with two morphologically different ovarian types

The purpose of this experiment was to determine the ovulation rate after treatment with human chorionic gonadotropin (hCG) in two groups of gilts characterized by different ovarian morphology: grape-type (GT; n = 11) and honeycomb-type (HT; n = 7). At 170 d of age (d 0), gilts were examined by laparoscopy and ovarian type was determined by the distribution of macroscopic follicles present on the ovarian surface. Five to ten minutes after surgery, each gilt received a single injection (i.m.) of 750 IU of hCG. At d 0, GT ovaries had a greater number of large follicles (greater than or equal to 6 mm) than HT ovaries (10.0 +/- .5 vs 2.6 +/- .3; P less than .05), whereas HT ovaries had more small follicles (1 to 3 mm; HT: 42.3 +/- .8 vs GT: 26.7 +/- .9; P less than .05) and total follicles (HT: 59.4 +/- 2.3 vs GT: 52.2 +/- 1.5; P less than .05), although numbers of medium follicles (4 to 5 mm) were similar (GT: 15.6 +/- .8 vs HT: 14.6 +/- 1.7; P greater than .10). Number of induced corpora lutea (CL) per ovary was greater (P less than .05) in gilts with GT ovaries (10.59 +/- 2.9 CL) than in gilts with HT ovaries (5.21 +/- .66 CL). Total weight of luteal tissue (LT) per ovary and serum progesterone concentrations 8 d after induction of ovulation were greater in GT gilts than in HT gilts (GT: 6.37 +/- 1.09 g vs HT: 3.31 +/- .49 g for LT, P less than .05; GT: 21.08 +/- 4.76 ng/ml vs HT: 13.40 +/- 2.05 ng/ml for progesterone, P less than .07).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell‐specific Distribution of Progesterone Receptors in the Bovine Ovary

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non‐pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down‐regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.     

Effect of the preovulatory LH surge on bovine follicular progesterone receptor mRNA expression

A growing body of evidence indicates that intrafollicular progesterone receptor signaling pathways are obligatory for follicle rupture. However, the intrafollicular localization and regulation of progesterone receptor expression during the periovulatory period in cattle are not known. In this study, we determined the effect of the preovulatory gonadotropin surge on localization and expression of progesterone receptor mRNA in bovine periovulatory follicular and luteal tissue. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 6, 12, 18, 24 (preovulatory follicles) and 48 h (CL) after a GnRH-induced LH surge (n=5-8 per timepoint). Expression of progesterone receptor mRNA was detected in periovulatory follicular and luteal tissue at all timepoints examined. Relative levels of progesterone receptor mRNA were dramatically upregulated within 6h after the LH surge compared to all other time points (P<0.0001). In situ hybridization analysis revealed that the significant increase in progesterone receptor mRNA expression was localized to the granulosal layer of preovulatory follicles. Our results indicate that progesterone receptor mRNA expression is upregulated specifically in the granulosal layer of bovine preovulatory follicles following the LH surge. Progesterone receptor signaling pathways may help mediate the effects of the preovulatory LH surge on follicle rupture in cattle.     

Detection of Follicular Apoptosis in Water Buffalo (Bubalus bubalis) Ovary by Histology and Nick End Labelling Technique

The objective of this experiment was to assess the features and extent of follicular apoptosis in the water buffalo (Bubalus bubalis) ovary using classical histology and nick end labelling technique. Ovaries (n = 40) procured from the slaughterhouse were used for the study. The sections (5 μm) were used for detection of terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labelling (TUNEL) and classical histology (H&E). Those follicles showing ≥ 5% TUNEL positivity (TUNEL assay) and pyknotic nuclei (histology) in granulosa cells were classified as atretic. Based on histology, the atretic primary and secondary follicles (%) were 93.82 and 95.62 respectively. The histology study reveals that the rates (%) of atresia in <1, 1–3, 3–5 mm and >5 mm were 36.90, 40.50, 62.84 and 74.5 respectively. Further the atretic tertiary follicles (%) were significantly lower than the primary and secondary classes of follicles. TUNEL assay reveals that the atretic rate (%) of tertiary follicles in <1, 1–3, 3–5 and ≥ 5 mm class follicles were 50.88, 53.84, 81.81 and 36.36 respectively. The percentage of atresia in >5 mm diameter follicles were significantly lower in TUNEL than histology. Percentages of granulosa and thecal cells positive for atresia by TUNEL were 30.7 ± 0.53 and 13.82 ± 0.18 respectively per follicle. The initial structural changes in atretic follicles were seen primarily in the granulosa cells. In severely atretic follicles TUNEL positive granulosa cells along with theca cells have to be considered in assessing the rate and extent of atresia.     

The follicular system of bovine ovaries after estrus synchronization using the luteolytic action of cloprostenol

Oestrus synchronization was studied in samples from six cows of the Black-Pied Lowland breed. Three cows four to five days from oestrus were used as the control; three animals with marked periodic corpora lutea were given an i. m. injection of 0.5 mg cloprostenol. The eighth day from the administration of the preparation, the ovaries of the cows were excised and, after histological processing in a simultaneous series in a 4mm interval, the preparations were subjected to qualitative and quantitative microscopic evaluation. The structure of non-atretic and atretic follicles was described in different stages of the atretic process. The lymphoid cells of atretic follicles were observed to penetrate into the granulosa membrane. A multiplication of non-atretic tertiary follicles was observed after the administration of cloprostenol. This multiplication was more pronounced on the right ovary where the preceding ovulation had taken place (P less than 0.01). The treated animals, compared with the controls, showed a significant multiplication of tertiary follicles at early atresia and at total collapse atresia (P less than 0.001), whereas the number of follicles with contractive atresia showed a significant decrease (P less than 0.001). The results suggest that cloprostenol can influence follicle population mostly through the stimulation of the growth and ripening of tertiary follicles; its modulation effect seems manifest itself in cooperating relation with gonadotrophic hormones, mainly with the follicular secondary hormone (FSH), in the theory of the complex effect of proteohormones .     

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.