- Email: [email protected]
Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles
Theriogenology39:421-432,1993
OXYTOCIN AND ESTRADIOL CONCENTRATIONS IN FOLLICULAR FLUID AS A MEANS FOR THE CLASSIFICATION OF LARGE BOVINE FOLLICLES R. Meidan,’ D. Wolfenson,’ W.W. Thatcher,2 E. Gilad, L. Aflalo,’ Y. Greber,’ E. Shoshani’ and E. Girsh’ ‘Department of Animal Science, Faculty of Agriculture The Hebrew University of Jerusalem, Rehovot 76100 Israel ‘Dairy Science Department, University of Florida Gainesville, Florida 32611-0691 Received for publication: January 28, 2992 Accepted: October 18, 1992 ABSTRACT Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F,a 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol 2 100 ng/ml and oxytocin < 65 pg/ml @ovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol< 100 ng/ml and oxytocin265 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol < 100 ng/ml and oxytocin < 65 pg/ml (atretic follicles). Treatment with prostaglandin F201 significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol:progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF,a was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles. Key words:
follicular fluid, bovine, estradiol, oxytocin, progesterone,
PGF*o
Acknowledgments We are grateful to Dr. A.P.F. Flint for providing oxytocin antiserum and to Dr. F. Kohen for providing estradiol and progesterone antisera. We are indebted to the employees of the Marbek abattoir and, especially, to Drs. Brenner and Hochman for their cooperation and assistance. Correspondence and reprints: R. Meidan, FAX #972-a-465763. This research was supported by BARD grant IS-1475-88.
Copyright
0 1993 Butterworth-Heinemann
Theriogenology
422
INTRODUCTION During the bovine estrous cycle, there are 2 or 3 waves of dominant follicle development (l-3). Dominant follicles present during early- or mid-luteal phases are normally non-ovulatory; however, they may become ovulatory if luteal function is disrupted (4). Hormonal concentrations in the follicular fluid of the bovine ovary fluctuate considerably with stage of the cycle, follicle size and follicle status (5-7). Steroid content in follicular fluid reflects the synthetic capabilities of the granulosa and thecal layers. Gonadotropins regulate androgen and estrogen secretion from theta and granulosa cells by inducing key biosynthetic enzymes (8). Gonadotropin-stimulated androgen secretion by the thecal layer provides the substrate for aromatase in granulosa cells, the level of which is stimulated by FSH (9,lO). Healthy large preovulatory follicles secrete high quantities of estradio1 (EJ and low quantities of progesterone (PJ, and are consequently characterized by a high F$:P4 ratio in the follicular fluid (6, 11-13). Ruminant ovarian granulosa cells also secrete oxytocin which may also be stored in the follicular fluid (14). Although there are numerous reports on the steroidal content of follicular fluid, only a few include a measurement of oxytocin. Data regarding oxytocin content of bovine preovulatory follicles are conflicting. Jungclas and Luck (15) could not detect oxytocin in the follicular fluid, whereas others have reported concentrations that range from small amounts to those as high as 1250 pg/ml (16, 17). Currently, follicles are classified as healthy or atretic according to the concentrations of different steroids in the follicular fluid; morphological criteria, which include vascularization of the thecal layer and the number of granulosa cells; consistency of follicular fluid; and status of the oocyte (6,18). Steroidal concentration of follicular fluids is the most convenient classification tool, and therefore it is widely used. However, studies have documented that the q:P4 ratio does not differentiate between atretic and healthy preovulatory follicles which are exposed to a gonadotropin surge (11,12). To oxytocin (PGF,(r), also was
accurately define the status of a follicle, we determined the concentration of in the follicular fluid as well as that of E, and Pa. The effect of prostaglandin F+Y administered randomly at 48 hours before slaughter, on follicular characteristics examined. MATERIALS
AND METHODS
Animals and Follicle Collection Large follicles (13 to 20 mm in diameter) from 109 cows and 17 heifers were collected 15 to 20 minutes after slaughter and were kept on ice until dissection. The largest follicle per pair of ovaries containing a regressed corpus luteum was selected. Thirty-one cows and 7 heifers were injected with PGF,a (25 mg Lutalyse per animal) 48 hours before Follicular fluid was slaughter. Follicular diameter was recorded using a micrometer. aspirated and frozen until assays for oxytocin, E, and P4 were performed. Granulosa cells
Theriogenology
423
were scraped from the follicular wall, as described by Meidan et al. (19)) and were counted. Cell viability was determined by trypan blue exclusion. An additional experiment was designed to test the effect of the gonadotropin surge (induced by GnRH injection) on hormone concentrations in the follicular fluid. The estrous cycle was synchronized with a control intravaginal drug release device (CIDR; containing 1.9 g Pa) that was inserted for 9 days and PGF,a! (25 mg) injected 2 days prior to removal of the CIDR device. Cows were assigned to 1 of 3 treatment groups. Treatment 1: on Day 6 of the estrous cycle, cows (n=4) were injected with PGF,or (25 mg), and ovaries were collected 36 hours later. Treatment 2: cows (n=3) were treated as in Treatment 1, and 24 hours after PGF,ol administration they were injected with a GnRH analog (Receptal; 10 pg per cow); ovaries were collected 13 hours later. Treatment 3: cows (n=3) were treated as in Treatment 2, with ovaries collected 23 hours after GnRH analog administration. Each pair of ovaries contained a regressed corpus luteum and a large follicle (1.2 to 1.6 cm). Follicular fluids were aspirated and kept frozen until assayed for oxytocin, E, and Pd. Reagents Biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO); [1,2,6,7- 3H] P4, and [2,4,6,7 3H] & were purchased from DuPont NEN Research Products (Boston, MA); PGF,a (Lutalyse) was purchased from the UpJohn Co. (Brussels, Belgium) and CIDR devices were acquired from Carter-Ho& Plastics Molding Co. (Hamilton, New Zealand). GnRH analog - Buserelin (Receptal) was purchased from Hoechst (Frankfurt, Germany). Antisera against steroids were generously provided by Dr. F. Kohen (Weizmann Institute of Science, Rehovot, Israel), while antiserum against oxytocin was kindly provided by Dr. A.P.F. Flint (The Zoological Society of London, Institute of Zoology, London, UK). Steroidal Content of Follicular Fluids Unextracted samples of follicular fluids were diluted (10 to 200 fold) in phosphate buffer. Progesterone and E, were assayed by radioimmunoassays as described previously (19). The sensitivity limits for E, and P4 were 2 and 7.8 pg/tube, respectively. The intraand inter-assay coefficients of variations were 12 and 11.6% for & and 4.5 and 5.2% for P,. Oxytocin Determination Oxytocin was measured by radioimmunoassay in an assay buffer consisting of 0.05M PBS, 0.2% gelatin and 2mM EDTA at a pH of 7.4; antiserum (0.2 ml of a 1:14,000 dilution) was added to 0.2 ml follicular fluid and incubated for 2 hours at 22°C. Iodination was carried out as described by Kasson et al. (20); iodinated peptide (4000 cpm) was added, and samples were incubated overnight at 22°C. The bound and free oxytocin was separated using a 1% charcoal, 0.1% dextran solution. Radioactivity of the supematant were also determined. Sensitivity of the assay was 1 pg/tube. Intra- and inter-assay coefficients of variation were 4.3 and 7%, respectively. Follicular fluids of several follicles were serially diluted and reassayed; the values obtained indicated good linearity of the assay.
Theriogenology
424
Data Analysis Follicles were separated into 2 distinct populations for both oxytocin and estradiol analyses. A cluster analysis was performed for the distributions of Q and a separate analysis for oxytocin using Fast Clus of Statistical Analysis System (21). Data were analyzed by the least squares analysis of variance using the General Linear Models procedure of the SAS (21). Separate analyses were performed for the following dependent variables: oxytocin, E,, P4, E$:P4, E,:oxytocin and for the number of granulosa cells. Sources of variance examined in the mathematical models were group (the 3 groups of follicles as described in the results section), PGF,cu-injected versus noninjected and their interaction. Correlation analysis was carried out for the different hormones in each group. The effect of PGF,ol on the distribution of follicles between follicular groups was examined by Chi-square analysis. Values for oxytocin, E-r, P4 in Treatments 1, 2 and 3 were analyzed by one-way analysis of variance. The values were considered significantly different at P50.05.
I 0
I 50
100
I/I
I
150 ’
200
Estracliol
Figure 1.
I 400
1 600
f 800
1 I 1000 120(
Ing/ml]
Estradiol and oxytocin concentrations in the follicular fluid of untreated or Each point represents an individual follicle. PGF,cr-treated animals. Where hormone content in different follicles overlapped only one symbol appears.
Theriogenology
RESULTS In the overall follicle population of this study, including the follicles from PGF,crtreated cows, there was no significant correlation (r=-0.16) between concentrations of oxytocin and Ez. The scattergram of I!$ vs oxytocin concentrations in the follicular fluid is shown in Figure 1. Cluster analyses were performed on I$ and oxytocin values. For E._,, the largest value for the lower cluster was 94.6 ng/ml, while the lowest value for the upper cluster was 104.5 ng/ml. For oxytocin the largest value for the lower cluster was 49.3 pg/ml, while the lowest value for the upper cluster was 65.0 pglml. These analyses separated ovarian follicles into 2 distinct populations, classified both by their E, and oxytocin values. Based on the cluster analyses described above, the limits chosen were 65 pg/ml for oxytocin and 100 ng/ml for E, (Figure 1). Consequently, 3 follicular groups were identified: Group I follicles (n=52, 41%) with %k 100 ng/ml and oxytocin< 65 pg/ml; Group II (n=19, 15%) with E, < 100 ng/ml and oxytocin 265 pg/ml, and Group III, obtained by cross classification (n =55, 44%) with E.r< 100 ng/ml and oxytocin < 65 pg/ml. The above was considered to be an objective method for partitioning follicle distributions for physiological analyses. Each group was then further subdivided into controls (untreated) and PGF,a-treated. The mean values of E, and oxytocin in the follicular fluid of the 3 groups of controls and PGF,a-treated cows are presented in Table 1. Follicular health status is reflected by E, and P4 concentrations in follicular fluids and in the number of viable granulosa cells within a follicle. Therefore, we next determined the values of these 2 independent variables (P4 concentrations and the number of granulosa cells) in the 3 groups (Table 2). The number of viable granulosa cells was significantly greater (P< 0.01) in Groups I and II than in Group III irrespective of PGF,(Y treatment; however, the number of granulosa cells and E, concentrations in Group I were significantly higher in follicles derived from cows injected with PGFzol 48 hours earlier (l?< 0.05 and P
426
Table 1.
Theriogenology
Hormonal concentrations of bovine follicles classified into 3 groups: Group I, estradiol> 100 ng/ml, oxytocin < 65 pglml; Group II estradiol< 100 ng/ml, oxytocin 2 65 pg/ml; Group III, estradiol < 100 ng/ml, oxytocin < 65 pg/ml Group I
II
III
Control
33 (37%)
14 (16%)
41 (47%)
PGF,cr-treated
19 (50%)
5 (13%)
14 (37%)
Total
52 (41%)
19 (15%)
55 (44%)
Control
397.6 +. 50.3
21.0 + 9.8
24.2 + 4.2
PGFzcr-treated
732.4 + 115.2*
6.4 + 3.0
33.4 + 8.5
Mean
519.0 +
16.7 + 7.1
26.5 +. 3.8
9.6 + 1.7
214.4 &- 41.6
15.0 * 2.1
10.4 -+ 3.2
359.4 +. 121.5
Number of follicles
Estradiol (ng/ml)
56.8
Oxytocin @g/ml) Control PGF,o-treated Mean
9.8 +. 1.5
252.6 +
44.8
9.6 t
1.8
13.6 + 1.7
Data are given as means f SEM. Numbers in parenthesis are the percentages of the total number of follicles. *Significantly different from the control (P <0.003). The differences observed in the hormonal composition of the follicles could have been due to the different timing of follicle collections in relation to the gonadotropin surge. Therefore, we examined the hormonal composition of large dominant follicles collected before the gonadotropins surge (36 hours after PGF,ar administration, Treatment l), or of follicles collected 13 and 23 hours after GnRH analog administration (Treatments 2 and 3, respectively). The results of this experiment are presented in Table 5. As expected, E, values (770.2 ng/ml) were high in the large dominant follicles in Treatment 1. Estradiol values dropped (P
Tberiogeno/ogy
Table 2.
427
Hormonal concentrations of bovine follicles classified into 3 groups: Group I, estradiolk 100 ng/ml, oxytocin < 65 pg/ml; Group II estradiol< 100 ng/ml, oxytocin ~65 pg/ml; Group III, estradiol< 100 ng/ml, oxytocin < 65 pg/ml Group I
Progesterone
II
III
(ng/ml)
Control
22.9 + 1.4
29.0 + 3.5
30.0 + 3.4
PGF,cr-treated
25.9 f
49.4 * 11.3*
53.9 f
Mean
24.0 + 1.4a
35.8 + 4.gb
37.3 + 6.4b
Control
4.1 +. 0.6 (12)
5.1 + 1.6 (4)
2.5 + 0.6 (14)
PGF,a-treated
6.1 + 0.8 (14)*
4.6 Ifi. 0.6 (3)
3.5 + 0.8 (12)
Mean
5.2 +. 0.5a
4.9 * l.oa
3.0 f
2.7
19.4
Viable granulosa cells x106
o.5b
Data are given as mean+SEM. Number in parenthesis is the number of follicles in which granulosa cells were counted. ~~gnilicantly different from the control (P < 0.05). ’ Mean values with different superscripts indicate a significant difference between groups. However, a significant (P < 0.03) increase in follicular oxytocin concentrations was observed at 23 hours after GnRH analog treatment (Table 5). Progesterone levels rose from 37.3 ng/ml in treatment 1 to 90.6 and 107.3 ng/ml (P
428
Theriogenology
and elevated P4 concentrations. Estradiol is the predominant hormone of the follicular phase, while P4 and oxytocin are prevalent after luteinization (14). The negative correlation which was found between & and oxytocin values in Group I and the positive correlation between oxytocin and P4 values found in Group II further support the characterization of Group I and Group II follicles as being pre-gonadotropin and post-gonadotropin surge, respectively. In addition to P4, oxytocin concentrations also were higher in Group II than in Group I. However, while P4 concentrations were only 15times higher, mean oxytocin levels in Group II were 25 times higher than in Group I. Our results clearly demonstrate that 23 hours after GnRH analog administration (Treatment 3), approximately 20 hours after the gonadotropin surge, follicular oxytocin concentration was indeed significantly higher than in follicles collected before the gonadotropin surge (Treatment 1) and in follicles harvested 13 hours after GnRH analog administration (Treatment 2, Table 5). Therefore, follicles in Treatment 3 were similar to Group II follicles. It should be noted, however, that the characterization of follicles as being either pre- or post-gonadotropin surge may not be absolute, due to the delay between the current synthetic abilities of follicular cells and hormone concentrations in follicular fluid. Table 3.
Correlation coefficients between hormonal concentration follicular fluid
in bovine
Oxytocin
Progesterone
Estradiol
r=-0.374”
r=0.026
Oxytocin
_____
r=O.Oll
Group I
Oxytocin
Progesterone
r=-0.046
r=0.261
Group II Estradiol Oxytocin
_____
r=0.661*
Group III Estradiol
r=0.041
Oxytocin
__-__
r=-0.231 r=O. 167
P
Theriogenology
429
that range from undetectable levels to 1250 pg/ml (U-17), collection in relation to the gonadotropin surge. Table 4.
reflect the timing of follicle
Hormonal ratios in bovine follicular fluid
Group I
27.1 4 4.0b
82.7 +. 9.9
Group II
0.71 f
0.2a
0.07 + 0.02
Group III
1.35 -
0.2a
3.20 +- 0.5
Data are the mean f SEM. - Estradiol (ng/ml); P4 - progesterone (ng/ml); OT - oxytocin @g/ml). “2, a, Different superscripts indicate significant difference between groups. The close relationship between follicular concentrations of E, and oxytocin are demonstrated both in the 3 follicular groups and also in the GnRH analog-treated cows. The period of 13 hours after the administration of GnRH analog was sufficient to significantly reduce E, concentrations; however, oxytocin levels remained low. The values of E, and oxytocin obtained from cows treated with the GnRH analog (Table 5) correspond well with the limits (100 ng/ml for E, and 65 pg/ml for oxytocin) obtained from the cluster analysis and which were used for follicle grouping. It was not until 23 hours post GnRH analog treatment that the increase in oxytocin (103.4 pg/ml) was observed, and E, concentrations further declined to values below 100 ng/ml (69.1 ng/ml). Table 5.
Treatment
Hormonal concentrations in large dominant follicles collected before the natural gonadotropin surge (Treatment I), or at 13 hours (Treatment 2) and 23 hours (Treatment 3) after administration of a GnRH analog Estradiol (ng/ml)
Oxytocin (pg/ml)
Progesterone
(ng/ml)
1
770.2 + 169.4a
22.5 + 10.la
37.3 +
4.6a
2
153.8 +. 35.7b
36.7 + 31.6ab
90.6 +
5.7b
69.1 -t_ 21.gb 103.4 + 31.3b 107.3 + 10.5b 3 D aY6 Different superscripts indicate a signifkant difference between groups. Progesterone concentration in Group III follicles were higher than in Group I preovulatory, pre-gonadotropin surge follicles, but were similar to Group II preovulatory, post-gonadotropin surge follicles. In fact, both E, and P4 concentrations in Group III follicles were similar to that of Group II follicles. However, Group III follicles had low oxytocin content and the smallest number of viable granulosa cells. It is therefore suggested that Group III follicles were comprised of aged, possibly atretic follicles. In our present study, a single large follicle per pair of ovaries (containing a regressed corpus luteum) was selected. As a result, Group III follicles probably had lost their dominance while a smaller, active follicle was already present in the ovary, a phenomenon which is frequently observed
Theriogenology
430
in ultrasonographic examinations of follicles (2,3). Thus, gross morphological examination of the ovaries is not sufficient for characterizing follicles that have been obtained randomly after slaughter. Overall, 44% of the follicles in our study fell into the atretic category. Currently, F&:P, ratios are often used to characterize follicles as healthy or atretic. This ratio is a diagnostic tool since P4 levels in atretic follicles are high but they are equally high in those classified as preovulatory post-gonadotropin surge follicles. Therefore, as also noted in earlier studies (6,11), the Fr:P4 ratio alone is not effective in differentiating between healthy, prwvulatory post-gonadotropin surge and atretic follicles. In contrast to Par oxytocin concentrations were only elevated in follicles exposed to the gonadotropin surge (Group II and Treatment 3). Oxytocin mean concentrations in the 3 groups of follicles ranged from 9.8 to 252.6 pg/ml. This suggests that not only is oxytocin specific to the gonadotropin surge, but it may also be more sensitive than P4, and may serve as an accurate tool for the classification of follicles. The fact that oxytocin (and not P4) is produced only by granulosa cells, as is &, may contribute to its specificity. Administration of PGFzol to cows and heifers induces lutwlysis from Day 6 to 16 of the estrous cycle (25). Reduction in P4 alleviates the negative feedback on gonadotropin secretion, which in turn supports follicular E, biosynthesis by several means: increased androgen biosynthesis, increased aromatase activity, and induced granulosa cell proliferation (4). Group I follicles of PGFzo-treated animals did indeed have a significantly higher Er content and more granulosa cells than follicles from untreated cows. This observation could be attributed to the timing of the follicular collection imposed by PGF,a! administration, which insures that these follicles are in synchronized advanced stages of development. Follicles of control animals were collected at various times during the follicular phase and stages of maturation. The percentage of healthy and atretic follicles was 53 and 47%, respectively, for control untreated animals, and 63 and 37%, respectively, for the PGF,otreated animals. This effect, although not statistically significant, could indicate that PGF,or may increase the occurrence of prwvulatory follicles as compared with the usual distribution of follicle types collected at slaughter. Prostaglandin F,a! may allow for the development of follicles which would otherwise regress in the presence of a high P4 concentration. In summary, we have demonstrated that a GnRH analog-induced gonadotropin surge alters not only q and P4 concentrations but also the oxytocin content of large dominant follicles. Analysis of follicular status is the objective of many in vivo studies, and it is also The important in the process of screening follicles for subsequent in vitro studies. classification method proposed herein, which is based on follicular fluid content of h and oxytocin, may efficiently serve these purposes.
REFERENCES 1.
Fortune, J.E., Sirois, J. and Quirk, S.M. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Theriogenology 29~95-109 (1988).
2.
O.J. Pierson, R.A. and Ginther, Theriogenology a:495-505 (1984).
Ultrasonography
of
the
bovine
ovary.
431
Theriogenology 3.
Savio, J.D.. , Keenan, L., Bol, M.P. and Roche, J.F. Pattern of growth of dominant follicles during the estrous cycle of heifers. Biol. Reprod. 83:663-671 (1988).
4.
Ireland, J.J. Control of follicular growth and development. (Suppl):39-54 (1987).
5.
Fortune, J.E. and Hansel, W. Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation. Biol. Reprod. 32:1069 1079 (1985).
6.
Kruip, Th.A.M. and Dieleman, S.J. Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and the stage of the estrous cycle. Theriogenology %:395-408 (1985).
7.
McNatty, K.P., Heath, D.A., Henderson, K.M., Lun, S., Hurst, P.R., Ellis, L.M., Montegomery, G-W., Morrison, L. and Thurley, D.C. Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary. J. Reprod. Fertil. z:39-53 (1984).
8.
Richards, J.S. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol. Rev. @:51-89 (1980).
9.
Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.H. Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrine Rev. 5:76-127 (1984).
10.
Skinner, M.K. and Osteen, K.G. Developmental and hormonal regulation of bovine granulosa cell function in preovulatory follicles. Endocrinol. 123: 1668-1675 (1988).
11.
Ireland, J.J. and Roche, J.F. Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles. J. Anim. Sci. a:157-167 (1983).
12.
Ireland, J.J. and Roche, J.F. Development of nonovulatory an&al follicles in heifers: changes in steroids in follicular fluids and receptors for gonadotropins. Endocrinology 112: 150-156 (1983).
13.
Dieleman, S.J., Kruip, Th.A.M., Funtijne, P., de Jong, W.H.R. and van der Weyden, G.C. Changes in estradiol progesterone and testosterone concentrations in follicular fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone. 3. Endocrinol. %:31-42 (1983).
14.
Schams, D. Luteal peptides and intercellular (Suppl.):87-99 (1987).
communication.
J. Reprod. Fertil. 3
J. Reprod. Fertil. 2
432
Theriogenology
15.
Jungclas, B. and Luck, M.R. Evidence for granulosa-theta interaction in the secretion of oxytocin by bovine ovarian tissue. J. Endocrinol. m:Rl-R4 (1986).
16.
Schams, D., Kruip, Th.A.M. and Koll, R. Oxytocin determination in steroid producing tissues and in vitro production in ovarian follicles. Acta Endocrinol. m:530-536 (1985).
17.
Schams, D., Walters, D.L., Schallenberger, Ovarian oxytocin in the cow. Acta Endocrinol.
18.
Spicer, L.J., Matton, P., Echtemkamp, S.E., Convey, E.M. and Tucker, H.A. Relationship between histological signs of atresia, steroids in follicular fluid, and gonadotropins binding in individual bovine antral follicles during postpartum anovulation. Biol. Reprod. 3&890-898 (1987).
19.
Meidan, R., Girsh, E., Blum, 0. and Aberdam, E. In vitro differentiation of bovine theta and granulosa cells into small and large luteal-like cells: morphological and functional characteristics. Biol. Reprod. Q:913-921 (1990).
20.
Kasson, B.G. Meidan, R. and Hsueh, A.J.W. Identification and characterization of arginine vasopressin-like substance in rat testis. J. Biol. Chem. m:5302-5307 (1985).
21.
SAS/SAT User Guide, Release 6.03 SAS Institute Inc. Cary, NC, 1988.
22.
Ivell, R., Brackett, K.H., Fields, M.J. and Richter, D. Ovulation triggers oxytocin gene expression in the bovine ovary. FEBS Lett. m:263-267 (1985).
23.
Meidan, R., Alstein, M. and Girsh, E. Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and IGF-I. Biol. Reprod. &:715-720 (1992).
24.
Voss, L.K. and Fortune, J.E. Oxytocin secretion by bovine granulosa cells: effect of stage of follicular development, gonadotropins and co-culture with theta intema. Endocrinology 128: 1991-1999 (1991).
25.
E., Bullermann, B. and Krag, H. 253 (Suppl.):147 abstr (1983).
Hansel, W. Physiology of the estrous cycle. J. Anim. Sci. 2 (Suppl.):404-423
(1983).
OXYTOCIN AND ESTRADIOL CONCENTRATIONS IN FOLLICULAR FLUID AS A MEANS FOR THE CLASSIFICATION OF LARGE BOVINE FOLLICLES R. Meidan,’ D. Wolfenson,’ W.W. Thatcher,2 E. Gilad, L. Aflalo,’ Y. Greber,’ E. Shoshani’ and E. Girsh’ ‘Department of Animal Science, Faculty of Agriculture The Hebrew University of Jerusalem, Rehovot 76100 Israel ‘Dairy Science Department, University of Florida Gainesville, Florida 32611-0691 Received for publication: January 28, 2992 Accepted: October 18, 1992 ABSTRACT Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F,a 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol 2 100 ng/ml and oxytocin < 65 pg/ml @ovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol< 100 ng/ml and oxytocin265 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol < 100 ng/ml and oxytocin < 65 pg/ml (atretic follicles). Treatment with prostaglandin F201 significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol:progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF,a was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles. Key words:
follicular fluid, bovine, estradiol, oxytocin, progesterone,
PGF*o
Acknowledgments We are grateful to Dr. A.P.F. Flint for providing oxytocin antiserum and to Dr. F. Kohen for providing estradiol and progesterone antisera. We are indebted to the employees of the Marbek abattoir and, especially, to Drs. Brenner and Hochman for their cooperation and assistance. Correspondence and reprints: R. Meidan, FAX #972-a-465763. This research was supported by BARD grant IS-1475-88.
Copyright
0 1993 Butterworth-Heinemann
Theriogenology
422
INTRODUCTION During the bovine estrous cycle, there are 2 or 3 waves of dominant follicle development (l-3). Dominant follicles present during early- or mid-luteal phases are normally non-ovulatory; however, they may become ovulatory if luteal function is disrupted (4). Hormonal concentrations in the follicular fluid of the bovine ovary fluctuate considerably with stage of the cycle, follicle size and follicle status (5-7). Steroid content in follicular fluid reflects the synthetic capabilities of the granulosa and thecal layers. Gonadotropins regulate androgen and estrogen secretion from theta and granulosa cells by inducing key biosynthetic enzymes (8). Gonadotropin-stimulated androgen secretion by the thecal layer provides the substrate for aromatase in granulosa cells, the level of which is stimulated by FSH (9,lO). Healthy large preovulatory follicles secrete high quantities of estradio1 (EJ and low quantities of progesterone (PJ, and are consequently characterized by a high F$:P4 ratio in the follicular fluid (6, 11-13). Ruminant ovarian granulosa cells also secrete oxytocin which may also be stored in the follicular fluid (14). Although there are numerous reports on the steroidal content of follicular fluid, only a few include a measurement of oxytocin. Data regarding oxytocin content of bovine preovulatory follicles are conflicting. Jungclas and Luck (15) could not detect oxytocin in the follicular fluid, whereas others have reported concentrations that range from small amounts to those as high as 1250 pg/ml (16, 17). Currently, follicles are classified as healthy or atretic according to the concentrations of different steroids in the follicular fluid; morphological criteria, which include vascularization of the thecal layer and the number of granulosa cells; consistency of follicular fluid; and status of the oocyte (6,18). Steroidal concentration of follicular fluids is the most convenient classification tool, and therefore it is widely used. However, studies have documented that the q:P4 ratio does not differentiate between atretic and healthy preovulatory follicles which are exposed to a gonadotropin surge (11,12). To oxytocin (PGF,(r), also was
accurately define the status of a follicle, we determined the concentration of in the follicular fluid as well as that of E, and Pa. The effect of prostaglandin F+Y administered randomly at 48 hours before slaughter, on follicular characteristics examined. MATERIALS
AND METHODS
Animals and Follicle Collection Large follicles (13 to 20 mm in diameter) from 109 cows and 17 heifers were collected 15 to 20 minutes after slaughter and were kept on ice until dissection. The largest follicle per pair of ovaries containing a regressed corpus luteum was selected. Thirty-one cows and 7 heifers were injected with PGF,a (25 mg Lutalyse per animal) 48 hours before Follicular fluid was slaughter. Follicular diameter was recorded using a micrometer. aspirated and frozen until assays for oxytocin, E, and P4 were performed. Granulosa cells
Theriogenology
423
were scraped from the follicular wall, as described by Meidan et al. (19)) and were counted. Cell viability was determined by trypan blue exclusion. An additional experiment was designed to test the effect of the gonadotropin surge (induced by GnRH injection) on hormone concentrations in the follicular fluid. The estrous cycle was synchronized with a control intravaginal drug release device (CIDR; containing 1.9 g Pa) that was inserted for 9 days and PGF,a! (25 mg) injected 2 days prior to removal of the CIDR device. Cows were assigned to 1 of 3 treatment groups. Treatment 1: on Day 6 of the estrous cycle, cows (n=4) were injected with PGF,or (25 mg), and ovaries were collected 36 hours later. Treatment 2: cows (n=3) were treated as in Treatment 1, and 24 hours after PGF,ol administration they were injected with a GnRH analog (Receptal; 10 pg per cow); ovaries were collected 13 hours later. Treatment 3: cows (n=3) were treated as in Treatment 2, with ovaries collected 23 hours after GnRH analog administration. Each pair of ovaries contained a regressed corpus luteum and a large follicle (1.2 to 1.6 cm). Follicular fluids were aspirated and kept frozen until assayed for oxytocin, E, and Pd. Reagents Biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO); [1,2,6,7- 3H] P4, and [2,4,6,7 3H] & were purchased from DuPont NEN Research Products (Boston, MA); PGF,a (Lutalyse) was purchased from the UpJohn Co. (Brussels, Belgium) and CIDR devices were acquired from Carter-Ho& Plastics Molding Co. (Hamilton, New Zealand). GnRH analog - Buserelin (Receptal) was purchased from Hoechst (Frankfurt, Germany). Antisera against steroids were generously provided by Dr. F. Kohen (Weizmann Institute of Science, Rehovot, Israel), while antiserum against oxytocin was kindly provided by Dr. A.P.F. Flint (The Zoological Society of London, Institute of Zoology, London, UK). Steroidal Content of Follicular Fluids Unextracted samples of follicular fluids were diluted (10 to 200 fold) in phosphate buffer. Progesterone and E, were assayed by radioimmunoassays as described previously (19). The sensitivity limits for E, and P4 were 2 and 7.8 pg/tube, respectively. The intraand inter-assay coefficients of variations were 12 and 11.6% for & and 4.5 and 5.2% for P,. Oxytocin Determination Oxytocin was measured by radioimmunoassay in an assay buffer consisting of 0.05M PBS, 0.2% gelatin and 2mM EDTA at a pH of 7.4; antiserum (0.2 ml of a 1:14,000 dilution) was added to 0.2 ml follicular fluid and incubated for 2 hours at 22°C. Iodination was carried out as described by Kasson et al. (20); iodinated peptide (4000 cpm) was added, and samples were incubated overnight at 22°C. The bound and free oxytocin was separated using a 1% charcoal, 0.1% dextran solution. Radioactivity of the supematant were also determined. Sensitivity of the assay was 1 pg/tube. Intra- and inter-assay coefficients of variation were 4.3 and 7%, respectively. Follicular fluids of several follicles were serially diluted and reassayed; the values obtained indicated good linearity of the assay.
Theriogenology
424
Data Analysis Follicles were separated into 2 distinct populations for both oxytocin and estradiol analyses. A cluster analysis was performed for the distributions of Q and a separate analysis for oxytocin using Fast Clus of Statistical Analysis System (21). Data were analyzed by the least squares analysis of variance using the General Linear Models procedure of the SAS (21). Separate analyses were performed for the following dependent variables: oxytocin, E,, P4, E$:P4, E,:oxytocin and for the number of granulosa cells. Sources of variance examined in the mathematical models were group (the 3 groups of follicles as described in the results section), PGF,cu-injected versus noninjected and their interaction. Correlation analysis was carried out for the different hormones in each group. The effect of PGF,ol on the distribution of follicles between follicular groups was examined by Chi-square analysis. Values for oxytocin, E-r, P4 in Treatments 1, 2 and 3 were analyzed by one-way analysis of variance. The values were considered significantly different at P50.05.
I 0
I 50
100
I/I
I
150 ’
200
Estracliol
Figure 1.
I 400
1 600
f 800
1 I 1000 120(
Ing/ml]
Estradiol and oxytocin concentrations in the follicular fluid of untreated or Each point represents an individual follicle. PGF,cr-treated animals. Where hormone content in different follicles overlapped only one symbol appears.
Theriogenology
RESULTS In the overall follicle population of this study, including the follicles from PGF,crtreated cows, there was no significant correlation (r=-0.16) between concentrations of oxytocin and Ez. The scattergram of I!$ vs oxytocin concentrations in the follicular fluid is shown in Figure 1. Cluster analyses were performed on I$ and oxytocin values. For E._,, the largest value for the lower cluster was 94.6 ng/ml, while the lowest value for the upper cluster was 104.5 ng/ml. For oxytocin the largest value for the lower cluster was 49.3 pg/ml, while the lowest value for the upper cluster was 65.0 pglml. These analyses separated ovarian follicles into 2 distinct populations, classified both by their E, and oxytocin values. Based on the cluster analyses described above, the limits chosen were 65 pg/ml for oxytocin and 100 ng/ml for E, (Figure 1). Consequently, 3 follicular groups were identified: Group I follicles (n=52, 41%) with %k 100 ng/ml and oxytocin< 65 pg/ml; Group II (n=19, 15%) with E, < 100 ng/ml and oxytocin 265 pg/ml, and Group III, obtained by cross classification (n =55, 44%) with E.r< 100 ng/ml and oxytocin < 65 pg/ml. The above was considered to be an objective method for partitioning follicle distributions for physiological analyses. Each group was then further subdivided into controls (untreated) and PGF,a-treated. The mean values of E, and oxytocin in the follicular fluid of the 3 groups of controls and PGF,a-treated cows are presented in Table 1. Follicular health status is reflected by E, and P4 concentrations in follicular fluids and in the number of viable granulosa cells within a follicle. Therefore, we next determined the values of these 2 independent variables (P4 concentrations and the number of granulosa cells) in the 3 groups (Table 2). The number of viable granulosa cells was significantly greater (P< 0.01) in Groups I and II than in Group III irrespective of PGF,(Y treatment; however, the number of granulosa cells and E, concentrations in Group I were significantly higher in follicles derived from cows injected with PGFzol 48 hours earlier (l?< 0.05 and P
426
Table 1.
Theriogenology
Hormonal concentrations of bovine follicles classified into 3 groups: Group I, estradiol> 100 ng/ml, oxytocin < 65 pglml; Group II estradiol< 100 ng/ml, oxytocin 2 65 pg/ml; Group III, estradiol < 100 ng/ml, oxytocin < 65 pg/ml Group I
II
III
Control
33 (37%)
14 (16%)
41 (47%)
PGF,cr-treated
19 (50%)
5 (13%)
14 (37%)
Total
52 (41%)
19 (15%)
55 (44%)
Control
397.6 +. 50.3
21.0 + 9.8
24.2 + 4.2
PGFzcr-treated
732.4 + 115.2*
6.4 + 3.0
33.4 + 8.5
Mean
519.0 +
16.7 + 7.1
26.5 +. 3.8
9.6 + 1.7
214.4 &- 41.6
15.0 * 2.1
10.4 -+ 3.2
359.4 +. 121.5
Number of follicles
Estradiol (ng/ml)
56.8
Oxytocin @g/ml) Control PGF,o-treated Mean
9.8 +. 1.5
252.6 +
44.8
9.6 t
1.8
13.6 + 1.7
Data are given as means f SEM. Numbers in parenthesis are the percentages of the total number of follicles. *Significantly different from the control (P <0.003). The differences observed in the hormonal composition of the follicles could have been due to the different timing of follicle collections in relation to the gonadotropin surge. Therefore, we examined the hormonal composition of large dominant follicles collected before the gonadotropins surge (36 hours after PGF,ar administration, Treatment l), or of follicles collected 13 and 23 hours after GnRH analog administration (Treatments 2 and 3, respectively). The results of this experiment are presented in Table 5. As expected, E, values (770.2 ng/ml) were high in the large dominant follicles in Treatment 1. Estradiol values dropped (P
Tberiogeno/ogy
Table 2.
427
Hormonal concentrations of bovine follicles classified into 3 groups: Group I, estradiolk 100 ng/ml, oxytocin < 65 pg/ml; Group II estradiol< 100 ng/ml, oxytocin ~65 pg/ml; Group III, estradiol< 100 ng/ml, oxytocin < 65 pg/ml Group I
Progesterone
II
III
(ng/ml)
Control
22.9 + 1.4
29.0 + 3.5
30.0 + 3.4
PGF,cr-treated
25.9 f
49.4 * 11.3*
53.9 f
Mean
24.0 + 1.4a
35.8 + 4.gb
37.3 + 6.4b
Control
4.1 +. 0.6 (12)
5.1 + 1.6 (4)
2.5 + 0.6 (14)
PGF,a-treated
6.1 + 0.8 (14)*
4.6 Ifi. 0.6 (3)
3.5 + 0.8 (12)
Mean
5.2 +. 0.5a
4.9 * l.oa
3.0 f
2.7
19.4
Viable granulosa cells x106
o.5b
Data are given as mean+SEM. Number in parenthesis is the number of follicles in which granulosa cells were counted. ~~gnilicantly different from the control (P < 0.05). ’ Mean values with different superscripts indicate a significant difference between groups. However, a significant (P < 0.03) increase in follicular oxytocin concentrations was observed at 23 hours after GnRH analog treatment (Table 5). Progesterone levels rose from 37.3 ng/ml in treatment 1 to 90.6 and 107.3 ng/ml (P
428
Theriogenology
and elevated P4 concentrations. Estradiol is the predominant hormone of the follicular phase, while P4 and oxytocin are prevalent after luteinization (14). The negative correlation which was found between & and oxytocin values in Group I and the positive correlation between oxytocin and P4 values found in Group II further support the characterization of Group I and Group II follicles as being pre-gonadotropin and post-gonadotropin surge, respectively. In addition to P4, oxytocin concentrations also were higher in Group II than in Group I. However, while P4 concentrations were only 15times higher, mean oxytocin levels in Group II were 25 times higher than in Group I. Our results clearly demonstrate that 23 hours after GnRH analog administration (Treatment 3), approximately 20 hours after the gonadotropin surge, follicular oxytocin concentration was indeed significantly higher than in follicles collected before the gonadotropin surge (Treatment 1) and in follicles harvested 13 hours after GnRH analog administration (Treatment 2, Table 5). Therefore, follicles in Treatment 3 were similar to Group II follicles. It should be noted, however, that the characterization of follicles as being either pre- or post-gonadotropin surge may not be absolute, due to the delay between the current synthetic abilities of follicular cells and hormone concentrations in follicular fluid. Table 3.
Correlation coefficients between hormonal concentration follicular fluid
in bovine
Oxytocin
Progesterone
Estradiol
r=-0.374”
r=0.026
Oxytocin
_____
r=O.Oll
Group I
Oxytocin
Progesterone
r=-0.046
r=0.261
Group II Estradiol Oxytocin
_____
r=0.661*
Group III Estradiol
r=0.041
Oxytocin
__-__
r=-0.231 r=O. 167
P
Theriogenology
429
that range from undetectable levels to 1250 pg/ml (U-17), collection in relation to the gonadotropin surge. Table 4.
reflect the timing of follicle
Hormonal ratios in bovine follicular fluid
Group I
27.1 4 4.0b
82.7 +. 9.9
Group II
0.71 f
0.2a
0.07 + 0.02
Group III
1.35 -
0.2a
3.20 +- 0.5
Data are the mean f SEM. - Estradiol (ng/ml); P4 - progesterone (ng/ml); OT - oxytocin @g/ml). “2, a, Different superscripts indicate significant difference between groups. The close relationship between follicular concentrations of E, and oxytocin are demonstrated both in the 3 follicular groups and also in the GnRH analog-treated cows. The period of 13 hours after the administration of GnRH analog was sufficient to significantly reduce E, concentrations; however, oxytocin levels remained low. The values of E, and oxytocin obtained from cows treated with the GnRH analog (Table 5) correspond well with the limits (100 ng/ml for E, and 65 pg/ml for oxytocin) obtained from the cluster analysis and which were used for follicle grouping. It was not until 23 hours post GnRH analog treatment that the increase in oxytocin (103.4 pg/ml) was observed, and E, concentrations further declined to values below 100 ng/ml (69.1 ng/ml). Table 5.
Treatment
Hormonal concentrations in large dominant follicles collected before the natural gonadotropin surge (Treatment I), or at 13 hours (Treatment 2) and 23 hours (Treatment 3) after administration of a GnRH analog Estradiol (ng/ml)
Oxytocin (pg/ml)
Progesterone
(ng/ml)
1
770.2 + 169.4a
22.5 + 10.la
37.3 +
4.6a
2
153.8 +. 35.7b
36.7 + 31.6ab
90.6 +
5.7b
69.1 -t_ 21.gb 103.4 + 31.3b 107.3 + 10.5b 3 D aY6 Different superscripts indicate a signifkant difference between groups. Progesterone concentration in Group III follicles were higher than in Group I preovulatory, pre-gonadotropin surge follicles, but were similar to Group II preovulatory, post-gonadotropin surge follicles. In fact, both E, and P4 concentrations in Group III follicles were similar to that of Group II follicles. However, Group III follicles had low oxytocin content and the smallest number of viable granulosa cells. It is therefore suggested that Group III follicles were comprised of aged, possibly atretic follicles. In our present study, a single large follicle per pair of ovaries (containing a regressed corpus luteum) was selected. As a result, Group III follicles probably had lost their dominance while a smaller, active follicle was already present in the ovary, a phenomenon which is frequently observed
Theriogenology
430
in ultrasonographic examinations of follicles (2,3). Thus, gross morphological examination of the ovaries is not sufficient for characterizing follicles that have been obtained randomly after slaughter. Overall, 44% of the follicles in our study fell into the atretic category. Currently, F&:P, ratios are often used to characterize follicles as healthy or atretic. This ratio is a diagnostic tool since P4 levels in atretic follicles are high but they are equally high in those classified as preovulatory post-gonadotropin surge follicles. Therefore, as also noted in earlier studies (6,11), the Fr:P4 ratio alone is not effective in differentiating between healthy, prwvulatory post-gonadotropin surge and atretic follicles. In contrast to Par oxytocin concentrations were only elevated in follicles exposed to the gonadotropin surge (Group II and Treatment 3). Oxytocin mean concentrations in the 3 groups of follicles ranged from 9.8 to 252.6 pg/ml. This suggests that not only is oxytocin specific to the gonadotropin surge, but it may also be more sensitive than P4, and may serve as an accurate tool for the classification of follicles. The fact that oxytocin (and not P4) is produced only by granulosa cells, as is &, may contribute to its specificity. Administration of PGFzol to cows and heifers induces lutwlysis from Day 6 to 16 of the estrous cycle (25). Reduction in P4 alleviates the negative feedback on gonadotropin secretion, which in turn supports follicular E, biosynthesis by several means: increased androgen biosynthesis, increased aromatase activity, and induced granulosa cell proliferation (4). Group I follicles of PGFzo-treated animals did indeed have a significantly higher Er content and more granulosa cells than follicles from untreated cows. This observation could be attributed to the timing of the follicular collection imposed by PGF,a! administration, which insures that these follicles are in synchronized advanced stages of development. Follicles of control animals were collected at various times during the follicular phase and stages of maturation. The percentage of healthy and atretic follicles was 53 and 47%, respectively, for control untreated animals, and 63 and 37%, respectively, for the PGF,otreated animals. This effect, although not statistically significant, could indicate that PGF,or may increase the occurrence of prwvulatory follicles as compared with the usual distribution of follicle types collected at slaughter. Prostaglandin F,a! may allow for the development of follicles which would otherwise regress in the presence of a high P4 concentration. In summary, we have demonstrated that a GnRH analog-induced gonadotropin surge alters not only q and P4 concentrations but also the oxytocin content of large dominant follicles. Analysis of follicular status is the objective of many in vivo studies, and it is also The important in the process of screening follicles for subsequent in vitro studies. classification method proposed herein, which is based on follicular fluid content of h and oxytocin, may efficiently serve these purposes.
REFERENCES 1.
Fortune, J.E., Sirois, J. and Quirk, S.M. The growth and differentiation of ovarian follicles during the bovine estrous cycle. Theriogenology 29~95-109 (1988).
2.
O.J. Pierson, R.A. and Ginther, Theriogenology a:495-505 (1984).
Ultrasonography
of
the
bovine
ovary.
431
Theriogenology 3.
Savio, J.D.. , Keenan, L., Bol, M.P. and Roche, J.F. Pattern of growth of dominant follicles during the estrous cycle of heifers. Biol. Reprod. 83:663-671 (1988).
4.
Ireland, J.J. Control of follicular growth and development. (Suppl):39-54 (1987).
5.
Fortune, J.E. and Hansel, W. Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation. Biol. Reprod. 32:1069 1079 (1985).
6.
Kruip, Th.A.M. and Dieleman, S.J. Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and the stage of the estrous cycle. Theriogenology %:395-408 (1985).
7.
McNatty, K.P., Heath, D.A., Henderson, K.M., Lun, S., Hurst, P.R., Ellis, L.M., Montegomery, G-W., Morrison, L. and Thurley, D.C. Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary. J. Reprod. Fertil. z:39-53 (1984).
8.
Richards, J.S. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol. Rev. @:51-89 (1980).
9.
Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.H. Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrine Rev. 5:76-127 (1984).
10.
Skinner, M.K. and Osteen, K.G. Developmental and hormonal regulation of bovine granulosa cell function in preovulatory follicles. Endocrinol. 123: 1668-1675 (1988).
11.
Ireland, J.J. and Roche, J.F. Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles. J. Anim. Sci. a:157-167 (1983).
12.
Ireland, J.J. and Roche, J.F. Development of nonovulatory an&al follicles in heifers: changes in steroids in follicular fluids and receptors for gonadotropins. Endocrinology 112: 150-156 (1983).
13.
Dieleman, S.J., Kruip, Th.A.M., Funtijne, P., de Jong, W.H.R. and van der Weyden, G.C. Changes in estradiol progesterone and testosterone concentrations in follicular fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone. 3. Endocrinol. %:31-42 (1983).
14.
Schams, D. Luteal peptides and intercellular (Suppl.):87-99 (1987).
communication.
J. Reprod. Fertil. 3
J. Reprod. Fertil. 2
432
Theriogenology
15.
Jungclas, B. and Luck, M.R. Evidence for granulosa-theta interaction in the secretion of oxytocin by bovine ovarian tissue. J. Endocrinol. m:Rl-R4 (1986).
16.
Schams, D., Kruip, Th.A.M. and Koll, R. Oxytocin determination in steroid producing tissues and in vitro production in ovarian follicles. Acta Endocrinol. m:530-536 (1985).
17.
Schams, D., Walters, D.L., Schallenberger, Ovarian oxytocin in the cow. Acta Endocrinol.
18.
Spicer, L.J., Matton, P., Echtemkamp, S.E., Convey, E.M. and Tucker, H.A. Relationship between histological signs of atresia, steroids in follicular fluid, and gonadotropins binding in individual bovine antral follicles during postpartum anovulation. Biol. Reprod. 3&890-898 (1987).
19.
Meidan, R., Girsh, E., Blum, 0. and Aberdam, E. In vitro differentiation of bovine theta and granulosa cells into small and large luteal-like cells: morphological and functional characteristics. Biol. Reprod. Q:913-921 (1990).
20.
Kasson, B.G. Meidan, R. and Hsueh, A.J.W. Identification and characterization of arginine vasopressin-like substance in rat testis. J. Biol. Chem. m:5302-5307 (1985).
21.
SAS/SAT User Guide, Release 6.03 SAS Institute Inc. Cary, NC, 1988.
22.
Ivell, R., Brackett, K.H., Fields, M.J. and Richter, D. Ovulation triggers oxytocin gene expression in the bovine ovary. FEBS Lett. m:263-267 (1985).
23.
Meidan, R., Alstein, M. and Girsh, E. Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and IGF-I. Biol. Reprod. &:715-720 (1992).
24.
Voss, L.K. and Fortune, J.E. Oxytocin secretion by bovine granulosa cells: effect of stage of follicular development, gonadotropins and co-culture with theta intema. Endocrinology 128: 1991-1999 (1991).
25.
E., Bullermann, B. and Krag, H. 253 (Suppl.):147 abstr (1983).
Hansel, W. Physiology of the estrous cycle. J. Anim. Sci. 2 (Suppl.):404-423
(1983).