Follicular growth and endocrine patterns of prepuberal heifers administered bovine follicular fluid and (or) follicle stimulating hormone

Animal Reproduction Science, 18 (1989) 227-242

227

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Follicular G r o w t h and E n d o c r i n e P a t t e r n s of P r e p u b e r a l Heifers A d m i n i s t e r e d B o v i n e Follicular Fluid and (or) Follicle S t i m u l a t i n g Hormone 3 M.T. MOSER, H.A. GARVERICK 4, M.F. SMITH 1 and R.S. YOUNGQUIST 2

Dairy Science Department, 1Animal Science Department and 2Department of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO 65211 (U.S.A.) 3Contribution from the Missouri Agri. Exp. Station Journal Series No. 10553, and NC-113 Regional Research Project, Methods for Improvement of Fertility in Cows Postpartum. 4Reprint requests should be addressed to: Dr. H.A. Garverick, 163 Animal Science Research Center, Dairy Science Department, University of Missouri, Columbia, MO 65211, U.S.A. (Accepted 14 September 1988)

ABSTRACT

Moser, M.T., Garverick, H.A., Smith, M.F. and Youngquist, R.S., 1989. Follicular growth and endocrine patterns of prepuberal heifers administered bovine follicular fluid and (or) follicle stimulating hormone. Anim. Reprod. Sci., 18: 227-242. Two experiments were conducted to determine if charcoal-extracted follicular fluid ( CFF ) from bovine ovaries inhibits FSH-induced follicular development in intact prepuberal heifers. In Exp. 1, thirty prepuberal heifers were assigned to saline, FSH or C F F / F S H treatment groups. In Exp. 2, thirty-two prepuberal heifers were assigned to four treatment groups (Saline, CFF, FSH, C F F / FSH) in a 2 × 2 factorial arrangement. In both studies, heifers were injected (i.v.) every 8 h for 88 h with CFF (8 ml, Exp. 1; 20 ml, Exp. 2) or saline. Follicle stimulatinghormone (3.3 mg with 14% LH, Exp. 1; 3.3 mg with < 1% LH, Exp. 2) was injected (i.m.) every 8 h starting 24 h after the initiation of CFF injections and continuing until termination of CFF administration. Plasma samples were collected via jugular venipuncture at 8 h intervals from just prior to (Exp. 1 ) or 48 h before (Exp. 2) CFF treatment until 96 h after, at which time both ovaries were removed. In both experiments total ovarian and follicular fluid weights increased (P < 0.05) following FSH treatment. However, total number of follicles was similar among treatments, FSH induced a shift (P<0.05) from small ( -<3 mm) to medium (7 to 9 ram) or large (10 to 13 ram) follicles. None of the above indices of FSH-induced follicular growth were affected (inhibited) by CFF. In summary, CFF did not inhibit FSH-induced follicular development in intact prepuberal heifers.

INTRODUCTION Administration of follicular fluid (FF) delays follicular maturation and return to estrus in cattle (Johnson and Smith, 1985; Quirk and Fortune, 1986), 0378-4320/89/$03.50

© 1989 Elsevier Science Publishers B.V.

228 sheep (McNeilly, 1984, 1985), pigs (Redmer et al., 1985 ) and horses (Bergfelt and Ginther, 1985). Although inhibition of preovulatory follicular development is clearly associated with decreased concentration of circulating FSH due to action of inhibin from the ovary (De Jong and Sharpe, 1976), FF may also inhibit folliculogenesis directly at the ovarian level (Quirk and Fortune, 1986 ) by the action of inhibin or other nonsteroidal factors present in FF (for review: Schwartz, 1982). Follicular fluid contains several nonsteroidal factors that may directly regulate the action of FSH. An FSH-binding inhibitor (FSH-BI) identified in bovine (Darga and Reichert, 1978; Fletcher et al., 1982) and porcine (Sluss and Reichert, 1984) FF may directly control folliculogenesis. In addition, a mitotic inhibitor (MI) has been found in ovine FF (Carson et al., 1986) and a follicle regulatory protein (FRP) in human (DiZerega et al., 1983a, b) and porcine (Kling et al., 1984) FF. Previous work from this laboratory with unilaterally ovariectomized (ULO) prepuberal heifers (Moser et al., 1989) suggests that charcoal-extracted FF (CFF) from the ovaries of cattle may not exert a direct effect on the ovary since FSH-induced follicular growth was not inhibited by injections of CFF. Two experiments using prepuberal intact heifers were designed to examine the effect(s) of greater concentrations of exogenous FSH and CFF on follicular growth. Bioassay of LH activity in the FSH preparations used in the previous study (Moser et al., 1989 ) and in Exp. 1 of this study revealed LH bioactivity (1.9 and 14%, respectively) in the FSH preparation. Therefore, in Exp. 2 a purified preparation of FSH was used to eliminate the possibility that LH contamination of the FSH preparation affected follicular development in prepuberal heifers. MATERIALSAND METHODS Experiment I

Thirty prepuberal Hereford heifers (B.W.-- 2+ SD; 159 + 24 kg) were randomly assigned to saline (n--10), FSH ( n = 1 0 ) and bovine, charcoal-extracted follicular fluid (CFF) and FSH (CFF/FSH; n = 10) treatments. One animal was removed from both the FSH and CFF/FSH treatment groups because ovulation occurred prior to ovariectomy as determined by presence of corpora lutea. On days 1 to 4 of the study, saline and FSH groups received 8 ml of saline intravenously every 8 h while the CFF/FSH group received 8 ml CFF every 8 h. In addition, 3.3 mg of FSH (in 1 ml saline) was given intramuscularly every 8 h (total dose 30 mg) to animals that were in the FSH and CFF/FSH groups, and saline (1 ml) was given to the remaining group days 2 to 4 of the experiment. The FSH used in this study was of porcine origin (BurnsBiotech Lab, Inc., Omaha, NE, FSH-P, Lot No's 588C84 and 592J84) and had biological activity of LH of 14% as determined by a rat intestitial cell bioassay

229

(Dufau et al., 1975). To assure exposure of the ovaries to CFF prior to FSH stimulation, injections of FSH were not started until 24 h following initiation of CFF treatment (0 h). Ovaries were removed from all heifers on day 4 (96 h). Peripheral plasma samples were collected prior to injections for determination of circulating concentrations of LH, FSH and estradiol-17fl (E2).

Experiment 2 Thirty-two prepuberal Angus heifers (183 + 40 kg) were randomly assigned to saline ( n = 8 ) , CFF ( n = 8 ) , FSH ( n = 8 ) and C F F / F S H ( n = 8 ) treatments as a 2 × 2 factorial design. One animal was removed from both the FSH and C F F / F S H treatment groups because ovulation had occurred prior to ovariectomy. Additionally, one animal was removed from the FSH group due to overstimulation (total ovarian weight > 28 g/ovary, more than three SD greater than the mean for treatment, 7 g/ovary), and another was removed from the C F F / F S H group because she was determined to be a freemartin at time of surgery. The protocol was similar to that of Exp. 1, except that 20 ml of CFF were used instead of 8 ml; a purified porcine FSH preparation (3.3 mg/injection, total dose 30 mg; L.E. Reichert, Jr., Albany Medical School, Albany, NY, p F S H 11-85-2) with less than 1% biological activity of LH was used; and peripheral plasma was collected at 4 h intervals for the first 20 h following CFF administration to more closely determine effect of CFF on concentration of endogenous FSH.

Preparation of charcoal-extracted follicular fluid Follicular fluid (FF) was obtained by aspirating bovine follicles ( < 20 mm diameter) collected at a local abattoir. The FF was kept on ice until centrifugation at 1700 × g for 1 h to remove cellular debris and stored at - 2 0 ° C. After a sufficient quantity of FF was collected to conduct the experiment, the FF was thawed and pooled. Norit A charcoal (10 m g / m l ) was added to the FF and mixed for 1 h at 4 ° C. Charcoal was removed from the FF by centrifugation for 20 min at 1700×g. The FF was again decanted and centrifuged at 30 000 × g for I h to remove remaining charcoal fragments. The CFF was frozen ( - 20 oC) until used in the experiment. This procedure was successful in preventing or removing bacterial contamination (at least Serratia liquifaciens) since negative results were obtained when the pools of CFF were cultured for the presence or absence of Serratia liquifaciens, a bacteria present in porcine FF that has been shown to secrete an FSH-BI (Sluss and Reichert, 1984). Concentrations of E2 (Kesler et al., 1977) and progesterone (P; Cantley et al., 1975) in the pooled CFF preparations prior to and after extraction were 54.0, 56.0 ng/ml and 0.2, 1.0 ng/ml, respectively for Exp. 1 and 68, 147 ng/ml and 0.3, 1.0 n g / ml for Exp. 2.

230

Blood collection and ovariectomy Blood samples were collected via jugular venipuncture into 20 ml heparinized tubes at 8 h intervals throughout the study beginning just prior to or 48 h before the first intravenous injections of CFF or saline (Exps. I and 2, respectively) and continuing until ovariectomy. Blood samples were drawn prior to intravenous and intramuscular injections. In addition, peripheral plasma was collected from heifers in Exp. 2 at 4 h intervals for the first 20 h following initiation of CFF injection. Plasma was stored at - 20 ° C until concentrations of FSH (Garverick et al., 1988), LH (Zaied et al., 1981 ) and E2 (Kesler et al., 1977) were measured. Gonadotropins were determined in a single assay for each study with intra-assay coefficients of variation of 9% and 2% for FSH and 7% and 7% for LH (Exps. 1 and 2, respectively). Intra-assay coefficients of variation for E2 were 9% for both studies and inter-assay coefficients of variation were 9% and 16% for Exps. 1 and 2, respectively. Prior to surgery, heifers were tranquilized with 10 mg of acepromazine maleate and locally anesthetized with 50 ml of 2% lidocaine hydrochloride. Ovaries were removed with an ecrasseur via a paralumbar incision using aseptic technique. After removal, ovaries were kept on ice and transported to the laboratory. Ovaries were weighed and diameter of surface follicles measured using calipers. Follicular fluid samples from follicles measuring 5 m m or greater were individually aspirated from one of the ovaries (determined at random). Within animals, FF for follicles < 5 m m was pooled. Concentrations of E2 (Kesler et al., 1977), P (Cantley et al., 1975) and T (Erb et al., 1976) in aspirated FF were determined. Intra-assay coefficients of variation for the FF steroids E2, P and T were 6%, 6% and 4% for Exp. 1 and 6%, 9% and 8% for Exp. 2, respectively. Inter-assay coefficients of variation were 9%, 7% and 3% for E2, P and T for Exp. 1 and 7%, 5% and 2% for Exp. 2. Ovaries (one per heifer) were sectioned into 0.5 mm slices, using a hand microtome and blotted dry and reweighed to determine FF weight (total ovarian weight - - blotted dry weight). Ovarian slices were lyophylized and reweighed. Total ovarian weights (g/ovary) for treatments were determined using both ovaries from each animal since no differences were detected between ovaries within animals. STATISTICALANALYSIS Statistical designs were similar between the two experiments. Experiment 1 was analyzed using a one way analysis of variance (ANOVA) to compare treatments (saline, FSH, and C F F / F S H ) . Experiment 2 was analyzed using a linear statistical model in a 2 × 2 factorial arrangement containing CFF and FSH (as the main effects) and their interaction. A completely randomized design ANOVA was used for non-time series parameters in these studies. Plasma hot-

231

mones which were measured over time were analyzed as a split-plot in time design (Gill and Hafs, 1971). The basic linear statistical model contained treatment, heifers within treatment, time and time × t r e a t m e n t interaction (treatment refers to CFF and FSH with respect to Exp. 2). The main plot effects of treatment was tested using heifers within treatment as the error term. Fisher's least significant different (LSD) procedure was used to determine mean differences (Cochran and Cox, 1957). Plasma E2 concentrations were different (P < 0.06) among groups prior to treatment in Exp. 2. Therefore, for each heifer the post-treatment concentrations of plasma E2 were adjusted by subtracting their mean pre-treatment E2 concentration from each post-treatment value. Split-plot analysis was performed using the adjusted E2 values. Follicular fluid steroids were analyzed by follicle size groups (small, <5; medium, 5 to 9; large, 10 to 13 m m ) . All ovarian parameters in Exp. 2 were analyzed in a 2 ><2 factorial arrangement with one exception. Steroids from large follicles (10 to 13 m m ) could only be compared between FSH and C F F / FSH groups since numbers of large follicles from saline and CFF groups were insufficient for comparison. Appropriate transformation of data was performed when variances of original data were heterogenous (Snedecor and Cochran, 1980). All analyses were preformed using least squares procedures for unequal subclass numbers (SAS, 1985). RESULTS

Experiment I FSH treatment increased (P < 0.05) ovarian and follicular fluid weights (Fig. 1), and CFF treatment did not block increases in ovarian and FF weight associated with FSH treatment (Fig. 1). Total number of surface follicles was not altered by exogenous FSH treatment, but number of small follicles ( < 3 m m ) decreased (P < 0.05) and number of larger follicles (7 to 9 m m and 10 to 13 m m ) increased (P < 0.05; Fig. 2). Follicular growth was stimulated in 88% of heifers (8/9) in the FSH treatment group. Administration of CFF did not inhibit the changes in follicular populations nor the stimulation of follicular growth (8/9 heifers responded to FSH in the C F F / F S H group ) associated with FSH treatment (Figs. 1 and 2). Plasma concentration of FSH declined 40% (P < 0.05) within 24 h following the first CFF injection (Fig. 3 ). Following administration of FSH, plasma concentrations of FSH increased ( P < 0 . 0 5 ) in the FSH and C F F / F S H groups. The rise in FSH was at least in part due to the crossreactivity (3%) of injected porcine FSH with bovine FSH antisera used in the radioimmunoassay. Concentration of plasma LH was similar among all treatment groups prior

232 m

Saline

[~FSH BCFF/FSH

5-

a

Ovarian Weight

Follicular Fluid Weight

Lyophilized Ovarian Weight

Fig. 1. Total, follicular fluid and lyophylized ovarian weights (least-square means of original data) in prepuberal heifers from Exp. 1 administered: saline (Control; n = 10), FSH (n--9) and charcoal-extracted bovine follicular fluid and FSH (CFF/FSH; n -- 9 ). Within each weight parameter, groups having different superscripts differ (abp < 0.05). Analysis performed after data was transformed (square root). 24-'1

I

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Total

m

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Follicular Oiameter (mm)

Fig. 2. Numbers of surface follicles per ovary (least-square means of original data) in prepuberal heifers from Exp. 1 following administration of saline (Control; n - - 1 0 ) , FSH ( n = 9 ) and charcoal-extracted bovine follicular fluid and FSH (CFF; n-- 9). Within each follicular diameter group, different superscripts differ (abp < 0.05 ). Analysis performed after data was transformed (square root).

233

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Fig. 3. Plasma concentrations (ng/ml; least-square means) of LH and FSH in prepuberal heifers from Exp. 1 administered saline (Control; n= 10), FSH (n=9) and charcoaJ-extracted bovine follicular fluid and FSH (CFF/FSH; n = 9). Standard errors; 0.21, 0.22, 0.22 ng/ml and 10.4, 1L0 and 11.0 ng/ml for L H and FSH, for saline, F S H and C F F / F S H treatments, respectively. TABLE 1 Intrafollicular concentrations (ng/ml) or estradiol- 17fl (E2), progesterone (P) and testosterone (T), for surface follicles less than 5, 5 to 9 and 10 to 13 mm in diameter from prepuberal heifers in Exp. 1 treated with saline, F S H or charcoal-extracted bovine follicular fluid (CFF) and F S H a Follicle size

Treatment

Ee

P

T

Small ( < 5 mm pooled/animal)

Saline ( n - - 8 b) FSH (n=4) CFF/FSH (n=4)

2.5_+0.4 3.1-+0.6 3.5-+0.6

30_+ 7 23-+10 12_+10

13 ± 3 d 1 +4 c 5 ± 4 cd

Medium (5to9mm)

Saline(n=24) FSH(n=69) CFF/FSH(n=63)

7 +5 c 17 ± 3 c 27 +3 d

21_+5 c 23_+3 cd 29+3 d

4 _+1d 1 ±1 c 4 -+1 d

Large (10to 13 mm)

Saline (n=15) FSH (n=51) CFF/FSH (n=33)

13 -+5 ¢ 20 + 3 c 26 + 4 d

41-+6 32_+3 32-+4

0.8_+0.7 c 1.7_+0.4 ~d 2.2_+0.5 d

aAll analysis performed after transformation (log) of original data. bn = minimum number of follicles used in determination of intrafollicular steroid concentration. c'dIf superscripts within a follicle size column differ ( P < 0 . 0 5 ) .

234

to the initiation of any treatments (Fig. 3). CFF treatment prior to that of FSH (first 24 h) did not affect mean LH concentration. Plasma LH concentration increased ( P < 0.05) following administration of FSH. This was most likely a result of the 14% LH contamination of the FSH preparation used in the study. Mean plasma E2 concentration was higher following exogenous FSH treatment ( P < 0.05) in animals that had received FSH alone or in combination with CFF compared to saline-treated heifers (7.7 ___1.2, 6.7 + 1.2 and 2.7 + 1.2 ng/ml, respectively). Follicular fluid concentrations of E2, P and T varied among treatments for small, medium and large sized follicles, but no consistent pattern of steroid concentration (E2, P and T) among treatments was observed (Table 1). In part, this may have been a result of high variability of steroids within treatments.

Experiment 2 Administration of exogenous FSH (main effect) in this study increased ( P < 0.05) ovarian and follicular fluid weights. T r e a t m e n t with CFF did not inhibit this response (Fig. 4). Number of total surface follicles was not changed by FSH treatment. However, number of small follicles ( < 3 m m ) was reduced ( P < 0 . 0 5 ) , and number of medium (7 to 9 m m ) and large (10 to 13 m m ) follicles was increased (P < 0.05; Fig. 5). Animals treated with CFF (main treatment) had lower ( P < 0.05 ) ovarian weights (4.7 + 0.48 and 3.2 + 0.48 g/ovary, absence and presence of CFF, respectively), but no other ovarian parameters were affected by CFF treatment. Treatment with FSH singly or in combination with CFF resulted in a 75% to 85% response to FSH stimulation (6/8 for FSH group, including one animal that ovulated and one that was overstimulated and removed from all further analyses; 6/7 for C F F / F S H group including one that ovulated). There were no interactions between the main effects for any ovarian parameters measured. Circulating concentrations of FSH and LH were similar among treatments during the pretreatment period (data not shown). Plasma FSH declined (P < 0.05) within 4 h following initiation of CFF treatment and was reduced by 50% or more of pretreatment concentration within 24 h (Fig. 6). Circulating FSH concentration remained lower ( P < 0.05 ) throughout the study in the CFF only treated group, but increased following exogenous FSH in C F F / F S H treated animals. The control (saline) group of animals had relatively constant levels of FSH throughout the duration of the study. FSH concentration apparently increased following administration of exogenous FSH but as previously mentioned, this was believed to be in part, the result of crossreactivity of the porcine FSH with the bovine FSH antisera used in the radioimmunoassay. The

235 [~1

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8-

b

CFF/FSH

7-

A o~

6-

50) 4a 3

a

Ovarian Weight

Follicular~Fluid Weight

Lyophylized Weight

Fig. 4. Total, follicularfluid and lypohylizedovarian weights (least-squaremeans of originaldata) in prepuberal heifers from Exp. 2 administered saline (Control; n = 8), charcoal-extractedbovine follicularfluid (CFF; n = 8 ), FSH (n = 6) and CFF/FSH (n = 6 ). Within each weightparameter, groups having different superscripts differ (,bp < 0.05). Analysisperformed after data was transformed (square root). increased concentration of FSH in latter stages of the experiment may have been the result of the natural rise in FSH normally associated with the LH surge. W h e n animals with only one or two consecutive samples of L H greater t h a n 5 n g / m l were removed from analysis (3 in the FSH and 4 in the C F F / F S H groups), the rise in plasma FSH was more gradual and concentration of FSH lower for those groups (data not shown). Concentration of LH in plasma was similar among all treatments prior to the initiation of exogenous FSH t r e a t m e n t (Fig. 6). CFF given in the absence of FSH did not affect LH concentration. The high LH levels in FSH and C F F / FSH treated animals after 72 h were probably primarily the result of LH surges ( > 5 n g / m l ) in some animals t h a t received exogenous FSH (previously described) and the slight increases before 72 h may have been caused by increased release in endogenous L H preceding the L H surge in these animals. W h e n animals exhibiting an L H surge (n= 7) were removed from the analysis, L H plasma concentrations did not exceed 4 n g / m l (data not shown). Circulating plasma E2 concentrations were not similar ( P < 0 . 0 6 ) among groups prior to t r e a t m e n t (3.8___0.5, 1.8___0.7, 4.1 _+0.8 and 0.7 + 0.1 n g / m l , means for saline, CFF, F S H and C F F / F S H groups, respectively). Therefore, adjusted data were used in the analysis. No differences were detected following

236

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a

5" 0

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7-9

10-14

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Fig. 5. Number of surface folliclesper ovary (least-square means of original data) in prepuberal heifers from Exp. 2 followingadministration of saline (Control; n = 8), charcoal-extractedbovine follicularfluid (CFF; n = 8 ), FSH (n = 6 ) and CFF/FSH (n = 6 ). Within each weightparameter, groups having different superscripts differ (abp< 0.05). Analysisperformedafter data was transformed (square root). t r e a t m e n t among the main effects or their interaction (5.2_+ 1.0, 2.2 _+0.6, 6.1 _+1.1 and 2.9 _+0.9 n g / m l unadjusted means for saline, CFF, F S H and C F F / F S H groups, respectively; overall adjusted mean 3.5 _+0.3 n g / m l ) . There were no main effects nor interactions of F S H or CFF on intrafollicular concentrations of E2, P and T in small and medium follicles, and intrafollicular steroids in small follicles ( < 5 ram) were similar among t r e a t m e n t s (Table 2). Progesterone concentration was higher in medium follicles (5 to 9 mm; P < 0.05 ) in CFF only treated animals, but there was no interaction. However, there was only a sample size of one in the CFF group and 2 in the saline group, but 67 in F S H and 48 in C F F / F S H groups. Main effects of F S H and CFF on large follicles (10 to 13 m m ) could not be examined since there were insufficient follicles in saline and CFF groups for analysis. One way analysis revealed higher (P < 0.05) E2 concentrations in the F S H group t h a n the C F F / F S H group (Table 2 ).

237 12-

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5

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CFF/FSH

Z ~

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Saline

80 ~

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Fig. 6. Plasma concentrations (least-square means; n g / m l ) of L H a n d F S H in prepuberal heifers from Exp. 2 administered saline (Control; n = 8), charcoal-extracted bovine follicular fluid (CFF; n=8), F S H ( n = 6 ) a n d C F F / F S H ( n - - 6 ) . S t a n d a r d errors; 0.12, 0.12, 0.14, 0.14 n g / m l a n d 3.7, 3.7, 4.3, 4.3 n g / m l for L H a n d FSH, for saline, CFF, FSH, a n d C F F / F S H , respectively. TABLE 2 Intrafollicular concentrations (ng/ml) of estrsdiol-17fl (E2) , progesterone (P) and testosterone (T) for surface follicles less than 5, 5 to 9 and 10 to 13 mm in diameter from prepuberal heifers in Exp. 2 treated with saline, charcoal-extracted bovine follicular fluid (CFF) and (or) FSH a Follicle size

Treatment

E2

P

Small ( < 5 mm pooled/animal)

Saline ( n = 7 b) CFF(n=4) FSH(n=6) CFF/FSH (n=4)

9± 10± 24± 14_+

Medium (5 to 9 ram)

Saline(n=2) CFF ( n = l ) FSH(n=67) CFF/FSH(n=48)

12±33 12_+57 52± 7 18± 8

Large (10 to 13 ram)

FSH ( n = 2 0 ) CFF/FSH ( n = l l )

89±17 d 17±22 ¢

5 6 5 6

21_+ 35_+ 25± 36±

T 8 8 9 9

11 1 16 3

_+4 ___5 ±4 ±5

31± 9 c 100_16 d 21± 2c 22_+ 2¢

2 0 6 2

±5 ±7 ±7 ±1

24_+ 3 24± 4

6.4±1.4 1.7±1.9

aAll analysisperformed aftertransformation (log) of originaldata. bn-- m i n i m u m number of folliclesused in determination of intrafollicularsteroidconcentration. c'dIfsuperscriptswithin a folliclesize column differ (P < 0.05).

238 DISCUSSION The inability of bovine charcoal-extracted follicular fluid (CFF) to inhibit FSH-induced follicular development of intact prepuberal heifers agrees with our previous work in ULO prepuberal heifers (Moser et al., 1989) and others in ewes (McNeilly, 1985). However, several aspects of our previous study (Moser et al., 1989) necessitated further investigation. In the previous study only 50% of the heifers responded to FSH treatment, therefore Exp. 1 was designed to determine if a larger dose (3.3 mg every 8 h; equivalent to the superovulatory dose for a cow) would result in a greater percentage of animals responding to FSH treatment. Data from Exps. 1 and 2 supported this premise (88% and 75% to 85%, respectively). In addition, these experiments examined the feasibility of the intact prepuberal heifer as a model for investigating the effects of CFF on FSH-induced follicular development. Ovarian responses to FSH and (or) CFF in these studies were similar to a previous study with ULO heifers. Previous work (Moser et al., 1989 ) agrees with the findings of Exp. 1 which suggest that exogenous FSH increases follicular growth which is not inhibited by CFF. Whether the observed increase in follicular growth in these studies was due solely to FSH or in part the effect of LH contamination of the FSH was unclear, therefore more highly purified FSH was used in Exp. 2. Results from Exp. 2 suggest the LH content of the FSH (14% in Exp. 1 ) did not influence the effects of FSH and (or) CFF since results were similar among experiments (Moser et al., 1989; Exps. 1 and 2 ). The FSH used in Exp. 2 contained minimal amount of LH ( < 1% ) but the amount was not believed to have been enough to influence ovarian follicular growth. Concentration of LH in plasma was not greatly increased following injection of FSH early in the experimental period. The elevated LH level in animals that received exogenous FSH near the end of the study was probably associated with a preovulatory rise in LH in some of the animals rather than LH in the FSH, and may account for the elevation of LH observed following FSH treatment in these experiments (Moser et al., 1989; Exps. i and 2). Suppression of circulating FSH by CFF is similar to studies using ULO heifers (Johnson et al., 1985; Moser et al., 1989), ULO gilts (Redmer et al., 1985) and intact ewes (McNeilly, 1985; Larson et al., 1987a). The reduction of FSH concentration but not LH following CFF treatment is also in agreement with the previous studies in ULO heifers (Johnson et al., 1985; Moser et al., 1989) and gilts (Redmer et al., 1985 ). In addition, the ability of CFF to reduce plasma FSH demonstrates that the injected CFF was biologically active. The effects of CFF and (or) FSH on circulating plasma E2 concentrations were inconsistent between the 2 experiments. However, E2 concentrations tend to increase following exogenous FSH (Exp. 1; Moser et al., 1989). The increase in ovarian and follicular fluid weights following exogenous FSH

239 was the result of a shift in size of antral surface follicles from small ( < 3 m m ) to larger (7 to 13 ram) follicles and not due to a change in total number of follicles. These observations (Exps. 1 and 2) agree with our previous work (Moser et al., 1989), which suggests stimulation of visible surface antral follicles or a rescue of atretic ones by FSH. Administration of bovine CFF fails to inhibit FSH-induced ovarian development in prepuberal heifers (present study, Moser et al., 1989) and ewes (McNeilly, 1985), suggesting that CFF does not exert a direct action at the ovarian level. Conversely, ovine CFF inhibits pregnant mare serum gonadotropin (PMSG)-induced ovarian growth in ewes (Cahill et al., 1985a, b). Administration of ovine CFF to PMSG-stimulated ewes with cauterized follicles reduced the number of follicles greater than 2 mm in surface diameter 3 days later when compared to animals administered only P M S G (Cahill et al., 1985a). In a subsequent experiment using hypophysectomized ewes, a lower growth rate of PMSG-stimulated follicles was observed in ewes that received ovine CFF, suggesting a direct ovarian action by CFF (Cahill et al., 1985b). Several studies in cattle (present study, Moser et al., 1989 ) and one in sheep (McNeilly, 1985) do not support the premise of a physiological action at the ovarian level by nonsteroidal factors such as FSH-BI (Fletcher et al., 1982), F R P (Kling et al., 1984), and MI (Carson et al., 1986) which have been demonstrated in vitro in follicular fluid of various species (bovine, porcine and ovine, respectively). Several factors may be associated with these results. The amount of these nonsteroidal factors injected into the heifers may have been insufficient, if present in the CFF, to exert an ovarian action. However, injections at 8 h intervals of 8 (Exp. 1 ), 10 (Moser et al., 1989) and 20 ml (Exp. 2) of CFF did not inhibit FSH-induced follicular development, but did reduce circulating concentrations of FSH. It is also possible that FSH flooded ovarian FSH receptors, thus preventing an action by FSH-BI on these receptors. However, FSH-BI is believed to be a non-competitive binding inhibitor (Reichert et al., 1982 ). Additional explanation for differences observed include a species variation between ovine and bovine FF. A direct ovarian action by CFF was suggested in studies involving ovine CFF (Cahill et al., 1985a, b). Studies in which bovine CFF was used (McNeilly, 1985; Moser et al., 1989; present study) do not support a direct ovarian action by CFF, except in one study using stalktransected ewes treated with bovine CFF (Larson et al., 1987b). Another difference among studies is that PMSG was used in studies where there was an effect of CFF (Cahill et al., 1985a, b), and FSH was used in studies where no effect of CFF on follicular development was observed (McNeilly, 1985; Moser et al., 1989, Exps. 1 and 2). Whether or not PMSG and FSH have similar actions at the ovarian level is unknown. Another plausible explanation is that FSH-BI (MW 5000; Fletcher et al., 1982) in CFF does not enter the follicle from the systemic circulation. However, studies suggest that substances with

240

molecular weights less t h a n 500 000 can pass freely through the basement membrane (Payer, 1973; Shagli et al., 1973; Andersen et al., 1976; Cran et al., 1976). Results from Exps. 1 and 2 suggest that CFF as administered in these experiments does not exert a localized action at the ovarian level on follicular sizes or intrafollicular steroid concentrations (E2, P and T ) in FSH-stimulated intact prepuberal heifers. Charcoal-extracted, bovine follicular fluid does, however, lower circulating concentration of FSH, supporting the concept of inhibin or inhibin-like substance in CFF suggesting t h a t the action(s) of FF on follicular growth is mediated by decreasing circulating concentrations of FSH and is not the result of a direct effect at the ovarian level. FSH-induced follicular growth is characterized by a shift in antral follicular populations from smaller to larger follicles. This induced follicular growth is believed to be due solely to the action of FSH and not the L H contamination in the FSH preparations. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Dr. Paul G. Harms, Dept. of Animal Science, Texas A&M University for measuring L H bioactivity in the FSH preparation used in this study; Dr. W.H. Fales, Vet. Med. Diagnostic Lab, for determining bacterial contamination in follicular fluid, and Dr. Gary Krause and Dr. Mark Ellersick, Agric. Exp. Station for statistical consultation; Sheila Ardrey for preparation of this manuscript; Dr. G.D. Niswender for supplying LH specific antisera; N I A D D K and N H H P , University of Maryland School of Medicine for supplying FSH specific antisera; Dr. N.R. Mason for estradiol17fl antisera; Dr. R.E. Short for progesterone antisera; Dr. S.A. Tillson for testosterone antisera; Dr. L.E. Reichert, Jr. for providing purified bovine LH reference standard and purified FSH for use in Exp. 2 and Dr. D.J. Bolt for supplying FSH reference standard.

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