influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on...

Influence of Stage of Cycle, Corpus Luteum Location, Follicle Size, and Number of Large Follicles on Estradiol-17/3 Concentrations in Bovine Follicles ~

S. A. B R A N T M E I E R , M. E. BELLIN, S. K. BOEHM, S. M. BUSHMEYER, C. L. KUBAJAK, M. R. DENTINE, R. R. G R U M M E R , and R. L. A X 2

Department of Dairy Science University of Wisconsin 1675 Observatory Drive

Madison 53706

ABSTRACT

Concentrations of estradiol-17/3 in follicular fluid were correlated to follicular size, stage of estrous cycle, location of corpus luteum, and presence of large follicles. Paired ovaries were obtained from 481 nonpregnant cows at slaughter and follicles were classified as ipsilateral or contralateral to the corpus luteum. Follicular fluid estradiol-1713 concentrations from 2494 small, 1485 medium, and 396 large follicles were quantified by radioimmunoassay. Stage of estrous cycle was estimated by visual examination of the corpus luteum. Follicles in stage 1 of the estrous cycle (d 1 to 4) had the highest estradiol-17/3 concentration and the smallest mean follicular diameter. Location of follicles relative to the corpus luteum had no influence on estradiol-17/3 concentrations. As follicular size increased, concentration of estradiol-17/3 also increased. The presence of a single large follicle did not affect the concentration of estradiol-1713 in medium or small follicles. In contrast, if multiple large follicles occurred in the same cow, concentrations of estradiol-17/3 were significantly lower in medium but not small follicles.

I N T R O D U C T I O N

The growing pool of bovine follicles is a very dynamic and unpredictable population. Unlike the human, where the preovulatory follicle is chosen 14 d before ovulation (10), the bovine follicle destined to ovulate cannot be deter- mined by size more than 3 d prior to estrus (7).

Most bovine follicles undergo atretic degen- eration rather than ovulation. To classify follicular health, histological and endocrine criteria have been employed. Ratios of estradiol- 17~ to progesterone are negatively correlated to numbers of pycnotic nuclei in granulosa cells in bovine follicles (14, 18). Estradiol-17/3 produc- tion by granulosa cells has been implicated for supporting oocyte maturation (17, 20), and estradiol-17/3 concentrations have been used as a sole determinant of atresia status (2, 3).

Previous studies have examined the concentrations of steroids in bovine follicles and their relationship to stage of the estrous cycle (11, 15), location of the corpus luteum (CL) (16), and follicular size (2, 11). This study was designed to examine the relationship between follicular size, stage of the estrous cycle, location of the CL, and influence of the largest follicle to estradio]-17/3 concentrations in individual follicles. We examined these factors in one model and statistically removed the influence of individual animals in comparison to other studies that employed limited numbers of animals and thus made interactions difficult to examine.

Received November 21, 1986. Accepted May 22, 1987. 1 Research was supported by the University of Wis-

consin College of Agricultural and Life Sciences and Hatch Grant 2778.

2 Reprint requests: 6 0 8 / 2 6 3 - 7 5 2 2 .

M A T E R I A L S A N D M E T H O D S

Follicles

Bovine ovaries from 481 nonpregnant cows were obtained from Champion Packers, Water-

1987 J Dairy Sci 70:2138-2144 2138

FOLLICULAR DEVELOPMENT IN COWS 2139

loo, WI wi th in 30 rain of slaughter. Stage of the estrous cycle was approx imated by visual inspect ion (13) o f the CL and was recorded as: stage 1 (d 1 to 4), stage 2 (d 5 to 10), stage 3 (d 11 to 17), or stage 4 (d t8 to 20) of the estrous cycle. Each foll icle was recorded for its loca t ion relative to the ovary with or wi thou t the most recent CL. External fol l icular d iameter f rom 4375 individual follicles was measured and fluid was aspirated f rom each foll icle using a 22- gauge needle and a 3-ml syringe. Only follicles f rom 3 to 20 m m were used. Fol l icular f luid was centr i fuged (9000 × g for 10 min) to remove granulosa cells, and the supernatants were f rozen in vials placed on dry ice and stored at - 2 0 ° C until assayed. Estradiol-17fi was assayed in fol l icular fluid in 2494 small (< 6 mm), 1485 med ium (6 to 10 mm), and 396 large follicles (11 to 20 ram).

Estradiol-17fl Radioimmunoassay Estradiol-17/3 was ext rac ted f rom 10 /21 o f

each foll icular f luid sample wi th .5 ml anes- the t ic grade diethyl ether. Tracer h o r m o n e was [2,4,6,16,17,-3H(N)] estradiol-17/3 (New En- gland Nuclear) with a specific activity o f 137 Ci /mmol . Ant isera specific for estradiol-17/~ were provided by Barry G. England and were previously character ized (8). Val idat ion o f the assay was previously repor ted (18). Addi- t ion of estradiol-17fl to fol l icular f luid demon- strated l inear i ty and parallelism with estradiol- 17/~ standards. Interassay and intraassay coeffi- cients of var ia t ion using foll icular fluid internal standards compu ted f rom 26 assays were 22.1 and 16.8%, respect ively, and the sensitivity was 17.6 -+ 1.5 pg/ml (mean + SEM, n = 6).

Assays were pe r fo rmed using 60,000 cpm of labeled steroid in each tube. Estradiol-17fl antisera were used at a final concent ra t ion o f 1:52,500 to bind approx imate ly 50% of the labeled steroid, and bound and free steroid were separated by the dext ran-coated charcoal technique. Superna tan t and 4.0 ml of 3a70B scinti l lat ion cocktai l (Research Products Inter- national) were added to miniscint i l la t ion vials and radioact iv i ty was counted in a Beckman liquid scinti l lat ion spec t ropho tomete r .

Statistics The Ranki t test (4) was per formed on

estradiol-17/3 concent ra t ions to test for normal- i ty of data. Data were t ransformed by convert-

ing concent ra t ions of estradiol-17/3 to natural logari thms [In (estradiol-17/3) + 11 to achieve normal i ty . Repor t ed means o f estradiol-17/3 were re t ransformed and represent the geometr ic means of the data unless stated otherwise.

Data were analyzed using the general l inear mode l procedure o f the Statistical Analysis Sys tem (22) with the fol lowing split-split plot statistical mode l :

Yijklmn = # + Si + Cj(Si) + CLk + (Si*CLk) + 01(C j) + F m + eijklmn

where:

Yijklmn = estradiol-17~ concent ra t ion or fol- l icular d iameter for the m th foll icle of jth cow at the ith stage of the cycle or the 1 th ovary;

/~ = c o m m o n mean; Si = f ixed effect of stage of estrous

cycle, i = 1 to 4, where 1 = d 1 to 4; 2 = d 5 to 10, 3 = d 11 to 17, a n d 4 = d 18 to 20 (ovulat ion = 0);

Cj = r andom effect associated within individual cow ;

CL k = f ixed ef fec t of locat ion based on posi t ion of CL, K = 1 was the ovary with the CL and K = 2 was the ovary no t possessing the CL;

Si*CL k = f ixed in teract ion of the i th stage of the cycle and the jth locat ion o f the CL;

0 1 ( C j ) = r a n d o m effect of the 1 th ovary wi th in each cow.

F m = f ixed effect of follicular size; depending upon the analysis, this was ei ther cont inuous or defined into three populat ions: small (<6 mm), med ium (6 to 10 mm), or large (11 to 20 mm) ;

eijklmn = r andom residuals; assumed indepen- dent and normal ly distr ibuted.

Individual mean differences were de termined by a t test af ter a significant F test (4).

Fol l icular size was omi t t ed f rom the model when fol l icular d iameter was used as the dependen t variable. In some models, the number of large follicles a cow possessed (ei ther 0, 1, or />2) was added to the split-plot as a main effect . In those instances, estradiol-17/~ concent ra t ions f rom large follicles were deleted f rom the data for the analysis.

Journal of Dairy Science Vol. 70, No. 10, 1987

2140 BRANTMEIER ET AL.

RESULTS AND DISCUSSION

Stage of Cycle

Characteristics of the population of follicles in relation to estimated stage of estrous cycle are shown in Table 1. Raw means of the data were not fitted in the appropriate model, so statistics could not be performed on the values. Use of the appropriate model showed that estradiol-17/~ concentration (Tables 2 and 3) and follicular diameter (Table 4) were related to stage of estrous cycle. Average follicular diameter was smaller in stage 1, but the overall average estradiol-17/3 concentration was higher compared with other stages (Table 1). Merz et al. (18) and Kruip and Dieleman (15) found that estradiol-17/~ concentrations were higher in proestrus (stage 4) and early lu- teal phase (stage 1). Ireland et al. (14) also found higher estradiol-17~3 concentrations post- ovulation (stage 1). From the study of Ireland et al. (12), it appeared that higher estradiol-17/3 concentrations may be due to one ovary; those researchers further speculated that only one follicle was involved. It is not known why there are greater concentrations of estradiol postovu- lation, but removal of inhibitory influences of the preovulatory follicle may have contributed to this increase.

Corpus Luteum and Corpus Luteum-Stage of Cycle Interaction

Concent ra t ions of estradiol-17/3 (Tables 2 = and 3) or follicular diameter (Table 4) were not significantly different when follicles located ipsilateral and contralateral to the CL were .E compared. Previous researchers showed similar results (16, 18). There was an interaction between location of the CL and stage of the ~. estrous cycle on follicular estradiol-17/3 con- o centrations (Tables 2 and 3) but not on follicu- "~ lar diameter (Table 4). The relative estimates of -~

e~

In estradiol-17/~ concent ra t ions used to examine CL and stage of cycle in terac t ion were calcu- lated by sett ing the estradiol-17fl concent ra t ion in stage 4 on the non-CL ovaries to 0 (Figure 1). The non-CL ovary had increased estradiol- '~ 17/3 concentrations compared to the CL ovary O in stages 1, 2, and 4. However, the reverse was ,.; seen in stage 3. The reason for the augmented u~ effect on the non-CL ovary is unknown. The CL would be producing substantial amounts of <

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TABLE 2. Analysis of variance for natural logarithm of estradiol-173 concentrations (ng/ml) in bovine follicular fluid using the split-split plot model.

Source df MS

Stage of cycle (S) 3 71.90"* Error (cow) 477 14.60 Corpus luteum location (CL) 1 5.24 CL × S 3 17.96" Error (ovary) 476 5.41 Follicle size 2 367.32** Error 3412 1.95

*Probability of significant F test with appropriate error term, P<-.05.

**Probability of significant F test with appropriate error term, P~<.01.

TABLE 3. Analysis of variance for natural logarithm of estradiol-17fl concentrations (ng/ml) with follicle size as a continuous variable using the split-split plot model.

Source df MS

Stage of cycle (S) 3 Error (cow) 476 Corpus luteum location (CL) 1 C L × S 3 Error (ovary) 469 Follicle size (FS) 1 FS × FS 1 Error 3363

76.52** 14.24

8.38 25.22*

5.32 729.74**

46.73** 1.92

*Probability of significant F test with appropriate error term, P<~.05. **Probability of significant F test with appropriate error term, P~<.01.

p roges t e rone dur ing stage 3 t h a t m ay be affecting the es t radio l -173 p r o d u c t i o n of the follicles o n t h a t ovary.

Follicular Diameter

Fol l icu la r d i a m e t e r was pa r t i t i oned in to t h r ee discre te size g roups [small ( < 6 mm) , m e d i u m (6 to 10 m m ) , or large (11 to 20 ram) ; Tab le 2] or was a l lowed to be a c o n t i n u o u s var iable (Table 3). In b o t h analyses, as fol l icular d i a m e t e r of size increased, es t rad io l -173 con- c en t r a t i ons increased. A p lo t of the m e a n estradiol-17fl c o n c e n t r a t i o n for each fol l icular d i a m e t e r is s h o w n in F igure 2. A s ta t is t ica l ly s igni f icant curva ture is seen in the u p w a r d d i rec t ion (R 2 = .95) ind ica t ing t h a t larger foll icles had increas ingly h igher c o n c e n t r a t i o n s o f h o r m o n e (Table 3). Small foll icles c o n t a i n e d 25% and m e d i u m 33% of the estradiol-17fl

c o n c e n t r a t i o n c o m p a r e d to large follicles (11 .93 ng /ml , g e o m e t r i c mean) . This is in a g r e e m e n t w i t h previous research which also

TABLE 4. Analysis of variance for surface diameter (mm) of bovine follicles using the split-split plot model.

Source df MS

Stage of cycle (S) 3 51.98" Error (cow) 477 15.58 Corpus luteum location (CL) 1 1.56 CL X S 3 19.99 Error (ovary) 476 9.13

*Probability of significant F test with appropriate error term, P~<.05.



2142 BRANTMEIER ET AL.

oLov ,R , NON*CL OVARy

STAGE OF ESTROUS CYCLE

Figure 1. Interaction of stage of the estrous cycle and location of the corpus luteum (CL) in relation to estradiol-17j3 concentration (* values relative to stage 4 of the contralateral ovary). In = Natural logarithm.

-1

Q:

W

y= ,5466 + .0966FS+ .0639FS*FS

10 20

FOLLICULAR SIZE (mm)

Figure 2. Relationship between follicular size (FS) and estradiol-17~ concentration. Data points are the geometric mean for each category of follicular size.

showed estradio1-17/3 concent ra t ions increased with increasing foll icular size (9, 11, 25).

In contrast to the overall relat ionship shown in Figure 2, estradiol-17/3 concent ra t ion rose sharply with respect to fol l icular size and then levels of f for each individual cow. The linear and quadra t ic terms for the average relat ionship f i t ted within a cow were .51 + .06 and - . 0 1 6 -+ .003. This wi th in-cow relat ionship is consistent with individual regulatory mechanisms enforc- ing a biological upper l imit to the amount of estradiol-17/3 that a given cow would produce f rom cells at larger foll icular sizes.

Data f rom follicles with very small (3 mm) and very large diameters (19 to 20 mm) were removed f rom analyses because o f insufficient numbers in those subclasses. Three-mil l imeter follicles had abnormal ly high estradiol-17/3 concent ra t ion (13.7 ng/ml), and al though we could no t account for it, we treated those follicles as outl iers and removed them f rom the analysis. Foll icles with a d iameter of 19 and 20 mm had very low mean estradiol-17~3 concentrat ions (<4 ng/ml) . Those follicles could be preovula tory follicles, since Dieleman and Blankenstein (5) have shown that preovulatory follicles start to produce progesterone after the LH surge. Al though estradiol-17~ and progesterone may be inversely related, increased progesterone p roduc t ion by itself does no t cause estradiol-17~ concent ra t ions to decrease. A second possibili ty is that they could have been small cysts that had poor estradiol secreting capabilities.

Effect of Large Follicles

Of all cows, 17.5% had two or more large follicles on their ovaries, but only 7.9% of the cows had two or more large follicles in stage 1 (Table 1). The recent removal o f a large follicle due to ovula t ion could account for that obser- vation. There is a high turnover of follicles th roughout the entire cycle, and the ovulatory follicle is no t de te rmined until a few days prior to estrus (7). Spicer and Ech te rnkamp (24) hypothes ized that growth rate o f small follicles is positively related to rate of atresia of large follicles as the estrous cycle progresses toward ovulat ion.

The number of large follicles had an effect on the rest of the follicular popula t ion (Table 5). Estradiol-17/3 concentra t ions were lower if a cow possessed two or more large follicles. There was an in terac t ion be tween the number of large follicles and foll icular size (Table 5, Figure 3). Concent ra t ions of estradiol-1713 in small follicles did not seem to be af fec ted by large follicles; however , if a cow possessed two or more large follicles, medium follicles exhibi ted lower estradiol-1713 concentrat ions . There were also reduced concent ra t ions o f estradiol-17/3 in large follicles if two or more large follicles occurred in the same cow. It appears that the estradiol-1713 concent ra t ions of small follicles are " p r o t e c t e d " whereas the large follicles exer ted a negative influence on estradiol-17/3 concent ra t ion in med ium follicles. Matron et al.



TABLE 5. Analysis of variance for natural logarithm of estradiol-17fl with the inclusion of an indicator variable for cows possessing O, 1, or/>2 large follicles.

Source df MS

Stage of cycle (S) 3 Error (cow) 473 Corpus luteum location (CL) 1 Large follicle (LF) 2 LF (EL) 2 SX LF 6 S X C L 3 S × LF (CL) 6 Error (ovary) 422 Follicle size (FS) 1 FS X CL 1 FS X LF 2 FS X CL X LF 2 Error 3059

68.26** 14.77

.47 122.38"*

7.02 5.196

23.61"* 4.13 3.30

284.93** 11.78"

122.75"* .63

1.61

*Probability of significant F test with appropriate error term, P~<.05.


(16) r epo r t ed tha t m e d i u m follicles are only i m p o r t a n t to the growing pool o f follicles if large follicles are rapidly turning over. Previous studies (15, 18) f o u n d two popula t ions o f large follicles, one wi th a high and one wi th a low estradiol-1713 concen t r a t ion in foll icular f luid. Thus, our observa t ion of reduced estradiol-17/3 concen t r a t ion in fluid o f mul t ip le large follicles would agree wi th those repor ts .

These data suggest tha t the large follicle possesses regulators of foll icular deve lopmen t . Those fac tors might include dif ferent ia l produc t ion of inhibin (21), a FSH-binding inhibi-

= oLo~oLL,oL,, 1 LG FOLLICLES 2. LG FOLLICLES

S U A t t M~OlUU L A ~ E

Figure 3. The geometric means of estradiol-1713 concentrations in small (<6 mm), medium (6 to 10 mm), and large (11 to 20 mm)follicles that contained no (0), one (1), or more than one (2+) large follicles.

tor (23), an aromatase inh ib i to r (6), insulin-like g rowth fac tors (1), and angiogenic fac tors (19). The cause-and-effect re la t ionship b e t w een those fac tors and the p roduc t i on of estradio1-17~ and their re la t ionship to foll icular select ion need fu r the r examina t ion .

REFERENCES

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2 Bellin, M. E., and R. L. Ax. 1984. Cbondroitin sulfate: An indicator of atresia in bovine follicles. Endocrinology 114:428.

3 Bellin, M. E., M. M. Hinshelwood, E. R. Hauser, and R. L. Ax. 1984. Influence of suckling and side of corpus luteum or pregnancy on folliculogenesis in postpartum cows. Biol. Reprod. 31:849.

4 Bliss, C. I. 1967. Statistics in biology. Vol. I. McGraw-Hill Book Co., New York, NY.

5 Dieleman, S. J., and D. M. Blankenstein. 1984. Changes in oestrogen-synthesizing ability of preovulatory bovine follicles relative to the peak of LH. J. Reprod. Fertil. 72:487.

6 diZerega, G. S., R. D. Marrs, P. C. Roche, J. D. Campeau, and O. R. Kling. 1983. Identification of protein in pooled human follicular fluid which suppress follicular response to gonadotropins. J. Clin. Endocr. Metab. 56:35.

7 Dufour, J., H. L. Whitmore, O. J. Ginther, and L. E. Casida. 1972. Identification of the ovulating follicle by its size on the different days of the estrous cycle in heifers. J. Anita. Sci. 34:85.


2 1 4 4 BRANTMEIER ET AL.

8 England, B. G., G. D. Niswender, and A. R. Midg- ley, Jr. 1974. Rad io immunoassay of estradiol-17/3 wi thou t chromatography. J. Clin. Endocr. Metab. 38:42.

9 Henderson, K. M., A. S. McNeilly, and I. A. Swan- ston. 1982. Gonadot ropin and steroid concentra- t ions in bovine follicular fluid and their relationship to follicle size. J. Reprod. Fertil. 65:467.

10 Hodgen, G. D., D. Kenigsburg, R. L. Collins, and R. S. Schenken. 1985. Selection of the dominan t ovarian follicle and hormona l enhancement of the natural cycle. Ann. New York Acad. Sci. 442:23.

11 Ireland, J. J., P. B. Coulson, and R. L. Murphee. 1979. Follicular development during four stages o f the estrous cycle of beef cattle. J. Anita . Sci. 49 :1261.

12 Ireland, J. J., R. L. Fogwell, W. D. Oxender , K. Ames, and J. L. Crowley. 1984. Product ion of estradiol by each ovary during the estrous cycle of cows. J. Anita. Sci. 59:764.

13 Ireland, J. J., R. L. Murphee, and P. B. Coulson. 1980. Accuracy of predict ing stages of bovine estrous cycle by gross appearance of the corpus lu teum. J. Dairy Sci. 63:155.

14 Ireland, J. J., and J. F. Roche. 1983. Growth and differentiat ion of large antral follicles after sponta- neous luteolysis in heifers: changes in concentra- t ion of ho rmones in follicular fluid and specific binding of gonadotropins to follicles. J. Anim. Sci. 57:157.

15 Kruip, T.A.M., and S. J. Dieleman~ 1985~ Steroid ho rmone concentra t ions in the fluid o f bovine follicles relative to size, quali ty and stage of oes t rous cycle. Theriogenology 24:395.

16 Matton, P., V. Adedlakoun, Y. Couture , and J. J. Dufour. 1981. Growth and replacement of the bovine ovarian follicles during the estrous cycle. J.

Anim. Sci. 52:813. 17 McGaughey, R. W. 1977. The culture of pig

oocytes in minimal med ium and the influence o f progesterone and estradiol-17~3 on meiotic maturation. Endocrinology 100:39.

18 Merz, E. A., E. R. Hauser, and B. C. England. 1981. Ovarian func t ion in the cycling cow: rela- t ionship between gonadot rop in binding to theca and granulosa and steroidogenesis in individual follicles. J. Anim. Sci. 52:1457.

19 Moor, R. M., and R. F. Seamark. 1986. Cell signal- ing, permeabil i ty and microvasculatory changes during antral follicle development in mammals . J. Dairy Sci. 69:927.

20 Moor, R. M., and A. O. Trounson . 1977. Hormonal and follicular factors affecting matura t ion o f sheep oocytes in vitro and their subsequent developmen- tal capacity. J. Reprod. Fertil. 49:101.

21 Radmanabhan , V. E., M. Convey, J. F. Roche, and J. J. Ireland. 1984. Changes in inhibin-like bioactiv- ity in ovulatory and atretic follicles and utero- ovarian venous blood after prostaglandin-induced luteolysis in heifers. Endocrinology 115 : 1332.

22 SAS Insti tute, Inc. 1986. SAS User's guide: statistics. SAS Inst., Inc., Cary, NC.

23 Sluss, P. M., P. W. Fletcher, and L. E. Reichert, Jr. 1983. Inhibit ion of ~2s I -human follicle-stimulating ho rmone binding to receptor by a low molecular weight fract ion of bovine follicular fluid inhibitor concentra t ion is related to biochemical parameters of follicular development . Biol. Reprod. 29:1105.

24 Spicer, L. J., and S. E. Echternkamp. 1986. Ovari- an follicular growth, func t ion and turnover in cattle: a review. J. Anim. Sci. 62:428.

25 Staigmiller, R. B., and B. G. England. 1982. Folliculogenesis in the bovine. Theriogenology 17:43.


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