Heterogeneity of gonadotropins and levels of uncombined luteinizing hormone subunits in pituitaries of cryptorchid rams.

Pituitaries were collected from intact rams and rams that had been rendered bilaterally cryptorchid by surgery to examine the effects of cryptorchidism on gonadotropin heterogeneity, levels of uncombined luteinizing hormone (LH) subunits, and the apparent molecular sizes of LH and follicle-stimulating hormone (FSH). Cryptorchid rams had higher pituitary contents of LH and FSH as well as reduced testicular weights. The levels of uncombined LH subunits, their apparent molecular weights, and the apparent molecular weights of intrapituitary LH were similar in control and cryptorchid rams. However, the apparent molecular weight of intrapituitary FSH was slightly larger in cryptorchid rams. Cryptorchidism altered the pattern of gonadotropin heterogeneity by shifting the distribution of LH isoforms towards basic components and shifting the distribution of FSH isoforms towards acidic components. Thus, it appears that the altered gonadal feedback mechanisms resulting from cryptorchidism modify the pattern of both LH and FSH heterogeneity by shifting the distribution of isoforms in opposite directions.

Effect of immunization against LHRH on isoforms of LH in the ovine pituitary.

The role of LHRH in modulating intrapituitary LH content as well as the distribution of LH among its isoforms was examined in sheep. Rams (n = 3) and wethers (n = 6) were actively immunized against an LHRH-human serum globulin conjugate. Pituitaries collected from these animals plus pituitaries from corresponding numbers of nonimmunized rams and wethers were extracted with a buffered saline solution containing protease inhibitors. Immunization markedly reduced total amounts of immunoreactive LH in the pituitary. An aliquot of each pituitary extract was desalted by flow dialysis against water and chromatofocused on a pH 10.5-7.0 gradient. Concentrations of LH in chromatofocusing fractions were determined by radioimmunoassay. LH in pituitary extracts resolved into nine peaks during chromatofocusing which were coded with letters beginning with the most basic isoform. The percentage of LH as the two most basic isoforms, A' and B, was similar (P > 0.05) in all treatment groups. Isoform H constituted a higher percentage (P < 0.05) of the LH in both castrate groups. Nonimmunized wethers had higher percentages of isoforms C, D and E (P < 0.05) and lower percentages (P < 0.05) of the acidic isoforms (coded as peak Z herein) than did other treatment groups. Thus, castration shifted the pattern of intrapituitary isohormones towards the more basic forms. Nonimmunized rams had a higher percentage (P < 0.05) of isoform G than did other groups. Isoform F, the most abundant isoform, was present as a higher percentage (P < 0.05) in immunized rams and wethers than in nonimmunized animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Bovine luteinizing hormone (LH) isoforms and amounts of messenger ribonucleic acid for alpha- and LH beta-subunits in pituitaries of cows immunized against LH-releasing hormone.

Ovariectomized beef cows were actively immunized against LHRH to test the hypothesis that decreased stimulation of gonadotropes would alter the distribution of LH isoforms and amounts of mRNA for subunits of LH in the pituitary. Eight long-term (3 yr) ovariectomized beef cows were randomly assigned to one of two treatments: immunization against LHRH conjugated to human serum globulin (n = 4) and nonimmunization (control, n = 4). Mean concentration of serum LH in cows immunized against LHRH (1.0 +/- .83 ng/ml) was less (p = 0.01) than in control cows (5.0 +/- 0.83 ng/ml). Amounts of alpha- (p = 0.13) and LH beta-subunit (p = 0.10) mRNA tended to be reduced in cows immunized against LHRH compared to control cows. However, weight of the anterior pituitary and concentrations of LH in this gland did not differ (p = 0.90) among cows from the two groups. Pituitary extracts were chromatofocused on pH 10.5-7.0 gradients, and concentrations of LH in eluent fractions were determined by RIA. Extracts of all pituitaries resolved into nine isoforms (designated A through H and Z beginning with the most basic form). Only isoform F (mid-alkaline elution, pH = 8.8) was influenced by treatment (p = 0.05). Cows immunized against LHRH had a greater relative amount of isoform F (42.1 +/- 1.4%) than controls (37.2 +/- 1.4%). In summary, immunization of cows against LHRH altered 1) circulating concentrations of LH, 2) amounts of mRNA for the subunits of LH, and 3) distribution of intrapituitary LH isoforms without changing the concentrations of LH in the anterior pituitary.

Effects of weaning on concentrations of inhibin in follicular fluid and plasma of sows.

Changes in plasma and follicular fluid concentrations of inhibin were examined in sows after weaning at 28-32 days post partum. From 0 to 48 h after weaning, inhibin concentrations were 200-300 times higher in follicular fluid from small (less than 4 mm) and medium-large (greater than or equal to 4 mm) follicles than in ovarian venous plasma. Inhibin concentrations increased in follicular fluid from medium-large follicles at 24 and 48 h after weaning; concentrations in ovarian venous plasma were positively correlated with the number of medium-large follicles (r = 0.40) and with ovarian venous plasma concentrations of oestradiol (r = 0.61). Blood samples were collected for 30 days from sows (n = 6) that exhibited oestrus within 5 days after weaning and from sows (n = 5) that remained anoestrous for 11 days after weaning. Plasma inhibin concentrations rose in oestrous and anoestrous sows by 12 h and continued to rise for 60 h after weaning. Plasma inhibin concentrations rose further and were higher at 3.5-4.5 days after weaning in oestrous sows than in sows that remained anoestrous. After oestrus, plasma inhibin concentrations declined. At weaning, plasma concentrations of follicle-stimulating hormone (FSH) were higher in sows that subsequently exhibited oestrus than in sows that remained anoestrous. After weaning, plasma concentrations of FSH declined in both groups, reached a nadir at 2.5 days, and increased gradually in anoestrous sows; oestrous sows exhibited an FSH surge at oestrus. Plasma FSH returned to preweaning concentrations in both groups of sows at Days 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of implanting bull calves with testosterone propionate, dihydrotestosterone propionate or oestradiol-17 beta prepubertally on the pituitary-testicular axis and on postpubertal social and sexual behaviour.

Twelve non-implanted crossbred bull calves served as controls and 30 crossbred bull calves (10/treatment) were implanted for 82 days, beginning at 34 days of age, to determine the influence of testosterone propionate (TP), dihydrotestosterone propionate (DHTP) and oestradiol-17 beta (E2) on prepubertal and pubertal pituitary-testicular function and on postpubertal social and sexual behaviour. Compared with control bulls, concentrations of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and inhibin concentrations were suppressed (P less than 0.01) in all implanted bulls. Testosterone (T) concentration increased (P less than 0.001) in TP-implanted, but decreased (P less than 0.01) in DHTP and E2 bulls during the implant period. LH response to gonadotrophin-releasing hormone (GnRH) challenge during the implant period (2.5 months of age) was less (P less than 0.01) in TP, E2 and DHTP bulls than in controls. A small but significant T response to GnRH occurred in control bulls at 2.5 months of age. LH and T responses to GnRH challenge at 7 months of age (100 days after implant removal) was similar (P greater than 0.20) in control and implanted bulls. Steroid implants administered prepubertally had no effect (P greater than 0.10) on postpubertal social and sexual behaviours, including number of flehmen responses, abortive mounts, services and competitive order score. Body weight did not differ (P greater than 0.10) between treatment groups, but testis size was reduced (P less than 0.01) during the implant period and up to 10 months of age in treated bulls compared with controls. Testes remained smaller in E2-treated bulls up to the end of the study (23 months of age), but daily sperm production and epididymal weight did not differ (P greater than 0.10) between treatment groups at slaughter. Control bulls reached puberty earlier (P less than 0.01; 270 +/- 11 days of age) than did TP (302 +/- 11 days), DHTP (309 +/- 11 days) or E2 (327 +/- 11 days) bulls. Although puberty was delayed in all implant groups, there was no difference in scrotal circumference at puberty (average 28.4 +/- 0.4 cm) between treatment groups. Our findings indicate that TP, DHTP and E2 implants administered prepubertally result in acute suppression of serum LH, FSH and inhibin during the implant period and in post-implant suppression of testis size and delayed puberty in bulls. The lack of treatment effect on behaviour suggests that steroidal programming of sexual behaviour occurs before 1 month of age in bulls.

Performance and carcass characteristics of bulls as influenced by exogenous hormones.

One-hundred and forty-four weanling bulls of Angus (A, n = 48), Simmental x Hereford (SH, n = 8) and Simmental (S, n = 48) breeding were either castrated, left intact, left intact and implanted with Ralgro or left intact and implanted with Synovex S. Cattle were slaughtered after 190, 246 or 315 days of high-energy feeding. The right side of each carcass was electrically stimulated. Steers were inferior to intact treatments for most performance and carcass cutability traits, but steers were superior in marbling and lean quality (P < 0·05). There were no differences (P>0·05) in dressing percentage or ribeye area per 100 kg of carcass weight among treatments. Relative to intact bulls, Ralgro and Synovex S increased carcass masculinity. Implanted intact treatments did not differ from nonimplanted intacts for feed conversion, average daily gain, yield grade characteristics, percent longissimus dorsi chemical fat and 9-10dash11th rib composition (P >0·05).

Palatability of beef from bulls administered exogenous hormones.

One-hundred and forty four weanling bulls of Angus (A, n = 48), Simmental x Hereford (SH, n = 48) and Simmental (S, n = 48) breeding were either castrated, left intact, left intact and implanted with Ralgro or left intact and implanted with Synovex S. Cattle were slaughtered after either 190, 246 or 315 days of high-energy feeding, and the right side of each carcass was electrically stimulated. Sensory analysis was conducted on longissimus dorsi steaks after 5 days aging and Warner-Bratzler shear force (WBS) was measured after 5, 10 and 15 days aging. Steers had lower (P < 0·05) WBS and more desirable sensory panel scores for juiciness, ease of fragmentation, amount of connective tissue and overall tenderness than all intact treatments. The implanting of weanling bulls with synthetic steroid hormone compounds did not make a practical improvement in beef tenderness.

Circulating concentrations of 17-estradiol influence pattern of LH in circulation of cows.

The objective of the research was to determine the relationship between circulating 17 beta-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E2 was influenced by interval between ovariectomy and the start of E2 treatment. Thirty-one nulliparous cows 3 yr of age were randomly assigned to a 2 x 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E2. The other eight cows served as non-implanted controls (n = 4-short term, n = 4-long term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-min intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-min intervals for an additional hour. Serum was analyzed for LH and E2. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrophin concentrations and ovulation rates in Suffolk ewes actively or passively immunized against inhibin alpha.

Mature Suffolk ewes were either actively or passively immunized against the synthetic fragment of porcine inhibin alpha, pI alpha(1-30), to determine the effects on gonadotrophin secretion and ovulation rate. Thirteen control ewes were immunized against human serum albumin, 12 ewes were actively immunized against pI alpha(1-30) and 36 ewes were passively immunized with pI alpha(1-30) antiserum. Blood samples were collected at 4-h intervals for 72 h from oestrus-synchronized ewes following the withdrawal of the progestagen pessaries. Mean gonadotrophin concentrations measured during the oestrous cycle of control ewes, ewes actively immunized against pI alpha(1-30) and ewes passively immunized against pI alpha(1-30) were similar, but their secretory profiles differed. Serum concentrations of follicle-stimulating hormone (FSH) were highest in ewes which had received antiserum at the time of pessary withdrawal; FSH concentrations did not decrease during the follicular phase of the oestrous cycle in ewes given antiserum 24 h after pessary withdrawal. Subtle but significant increments in serum FSH concentrations were observed in all passively immunized ewes in which sampling commenced at the time of treatment. The amplitude of the preovulatory luteinizing hormone (LH) peak, but not of the FSH peak, and the postovulatory secondary rise in FSH were lower (P less than 0.05) in actively immunized ewes than in control ewes. The mean (+/- s.e.) ovulation rate for actively immunized ewes (6.6 +/- 1.0) was 3 times higher (P less than 0.05) than that for control ewes (2.0 +/- 0.2), but was unaffected by passive immunization (range, 1.8-2.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Ovine luteinizing hormone. V. Significance of flow-through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application.

In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.

Ovarian follicle populations and ovulation rates of Finnish Landrace cross ewes in different nutritional states and associated profiles of gonadotrophins, inhibin, growth hormone(GH) and insulin-like growth factor-I.

Effects of ewe body condition and level of feed intake on ovarian follicle populations and ovulation rates were studied in three groups of 12 to 15 Finnish Landrace cross ewes. Ewes were fed to achieve target body condition scores of either 2.0 (low, LM) or 2.5 (moderate, MM) and then fed a complete pelleted ration for three weeks before study so that they maintained liveweight. Ewes of a third group were also fed to achieve condition scores of 2.5 and then fed ad libitum (MAL). Neither ewe body condition nor level of feed intake significantly affected either the number of large follicles (greater than or equal to 4 mm diameter) or the ovulation rate. The proportion of estrogenic follicles was lower in MM than LM ewes (0.77 vs. 0.96; P less than 0.05), but there was no difference in this proportion between MAL and MM ewes. Insulin-like growth factor-I (IGF-I) concentrations in the follicular fluid were unaffected by ewe body condition or level of feed intake despite significant treatment differences in circulating concentrations. Inhibin concentrations were higher in estrogenic follicles of LM compared with MM ewes, and this difference was reflected in circulating profiles. Treatment differences in LH profiles were not associated with any difference in follicle populations or ovulation rate. There were no consistent treatment effects on FSH concentrations. It is concluded that the roles of inhibin and IGF-I in the control of follicle development cannot be adequately assessed on the basis of circulating concentrations alone and that there is a need to measure intrafollicular hormone profiles and associated effects on follicle physiology.

Prepubertal changes in plasma FSH and inhibin in Holstein bull calves: responses to castration and(or) estradiol.

The objectives of two studies were to determine 1) whether plasma concentrations of inhibin (INH) changed with age in prepubertal bulls and whether these changes were related to changes in FSH, testosterone or testis length; and 2) whether castration and(or) estradiol implants affected plasma concentrations of INH and FSH. In Exp. 1, plasma INH remained constant from 4 until 8 wk of age then increased from 120 pM to 202 pM between 8 and 12 wk. Thereafter, INH decreased to 90 pM by 36 wk. Between 4 and 10 wk, plasma FSH increased from .32 to .43 ng/ml, apparently increasing before the initial rise in plasma INH. Between 10 and 12 wk, FSH declined from .43 to .33 ng/ml. After 12 wk, FSH increased as INH decreased. Initial increases in testis length and concentrations of plasma testosterone occurred at 14 wk coincident with the second rise in FSH. In Exp. 2, bull calves were either left intact, castrated, castrated and implanted with estradiol, or left intact and implanted with estradiol at 7.5 wk of age. Castration decreased concentrations of INH and increased concentrations of FSH. Castrated calves implanted with estradiol had decreased concentrations of both INH and FSH. Intact bulls implanted with estradiol had decreased concentrations of FSH relative to intact unimplanted bulls; however, concentrations of INH did not display the age-related changes observed in intact, unimplanted bulls. In summary, age-related changes in plasma INH and FSH occur in bulls. Furthermore, plasma concentrations of INH and FSH increased before changes in gonadal size were detected. The bovine testis may be a major source of circulating INH because castration decreased concentrations of plasma INH.

Pituitary and testicular responses of beef bulls to active immunization against inhibin alpha.

Prepubertal crossbred beef bulls served as controls or were actively immunized against the N-terminal, 30-amino acid synthetic fragment of porcine inhibin alpha, pI alpha (1-30). Antibody titers were detected in sera (greater than 40% B/BO in sera diluted 1,000-fold) but not in rete testis fluid of 390-d-old bulls. Serum FSH and inhibin remained static during a 5-h intensive bleed; inhibin was not acutely affected by a 15-fold LH rise and a threefold FSH rise induced by exogenous GnRH. Serum FSH, but not LH or testosterone, was consistently elevated (P less than .05) in immunized bulls compared with control bulls. Neither pituitary weight, pituitary gonadotropin content nor pituitary FSH/LH ratios were affected (P greater than .10) by pI alpha(1-30) active immunization. Testicular sperm density was greater (60 x 10(6) vs 45 x 10(6) sperm/g testis; P less than .10) in immunized bulls, but testes weight, epididymides weight and total daily sperm production remained unchanged. These results suggest that inhibin is important for regulation of FSH secretion and testicular function. Immunization with suitable inhibin vaccines may improve bull fertility.

Intramuscular collagen and serum hydroxyproline as related to implanted testosterone, dihydrotestosterone and estradiol-17 beta in growing wethers.

Relationships of implanted testosterone, dihydrotestosterone and estradiol-17 beta to collagen degradation and intramuscular collagen concentration and stability were determined. Intramuscular collagen content, solubility and shrinkage temperature and serum hydroxyproline were analyzed in groups of six rams, wethers, and wethers implanted with various levels of testosterone or dihydrotestosterone and groups of 10 rams, wethers and wethers implanted with estradiol-17 beta, dihydrotestosterone or a combination of these two steroids. Intramuscular collagen content in both experiments was higher (P less than .05) in muscles of rams than in muscles of wethers. Administration of the highest level of testosterone to wethers raised (P less than .05) total and insoluble intramuscular collagen to concentrations noted in rams. Administration of the testosterone metabolite, dihydrotestosterone, to wethers had no effect on intramuscular collagen. Administration of estradiol-17 beta to wethers tended to raise concentrations of intramuscular collagen so that they were no longer lower (P less than .05) than those in rams. Collagen stability as measured by solubility and thermal shrinkage temperature did not differ among rams, wethers or implanted wethers (P greater than .05). Increases in collagen accretion due to hormone administration were observed to be the result of increases in the insoluble portion of the intramuscular collagen (P less than .05).

Growth hormone and insulin-like growth factor-I responses in steers actively immunized against somatostatin or growth hormone-releasing factor.

Steers were actively immunized at 81 days of age against human serum albumin (hSA; controls) or hSA conjugated to either somatostatin (SRIF) or growth hormone-releasing factor (GRF). Binding titres were observed for the respective peptide antigens after all steers had been given booster immunizations. Although no effects of treatment were observed in SRIF-immunized steers, mean serum concentrations of GH and insulin-like growth factor (IGF-I) were suppressed (P less than 0.01) in GRF-immunized steers when compared with hSA-immunized controls. Mean concentrations of prolactin did not differ with treatment but showed seasonal fluctuations (P less than 0.001) associated with changes in the daylength. In contrast to its marked effect upon serum concentrations of IGF-I, immunization against GRF resulted in a relatively small (6%) but significant decrease in body weight gain (P less than 0.01) and an increase in carcass backfat thickness (P less than 0.05). In summary, our findings have shown the susceptibility of steers to growth modulation by GRF immunoneutralization. Secondly, the poor relationship observed between serum concentrations of IGF-I and growth rates in GRF-immunized steers suggested that circulating IGF-I may not be the principle factor determining the post-weaning growth rate in cattle.

Enrichment of bovine X- and Y-chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter.

Bovine spermatozoa were assessed indirectly for the presence of a Y chromosome by monitoring expression of the H-Y antigen. Spermatozoa labeled with a monoclonal H-Y antibody (MoAb) and fluorescein-conjugated goat antibody to mouse F(ab)2 were counted with both a fluorescent microscope and a fluorescence-activated cell sorter (FACS). Of ejaculated spermatozoa, 40% to 60% fluoresced by this procedure compared to 1% to 15% of sperm reacted with nonimmune serum. Semen from three bulls was exposed to nonimmune serum (control) or MoAb, sorted by FACS, and analyzed for DNA content with a scanning microdensitometer. Control samples showed two distinct peaks with a mean difference in DNA content of 3.95%; these peaks were assumed to represent Y- and X-chromosome-bearing spermatozoa populations, respectively. DNA analyses of the MoAb-treated sperm of three bulls that sorted positively for H-Y antigen (fluorescent sperm) yielded ratios of Y- to X-chromosome-bearing spermatozoa of 76 : 24, 88 : 12, and 77 : 23, and those sorted negatively for H-Y antigen (nonfluorescent sperm) yielded ratios of 26 : 74, 35 : 65, and 23 : 77. The proportions of Y- and X-chromosome-bearing spermatozoa in nonsorted samples were not different from 50 : 50. Suitable MoAbs can be used in conjunction with FACS to enrich the proportion of Y- or X-chromosome-bearing spermatozoa in bovine semen.

Immune, growth and carcass responses of ram lambs to active immunization against desulfated cholecystokinin (CCK-8).

This study explored feed intake and carcass responses to active immunization against desulfated cholecystokinin-octapeptide (CCK-8) in ram lambs. Antibody titers 8 wk following primary immunization and booster immunizations given at 4 and 6 wk averaged greater than 1:1,000. Titers increased to greater than 1:10,000 by 16 wk following a final booster immunization at 11 wk. The antibodies developed against desulfated CCK-8 exhibited 29% and 13% cross-reactivities for sulfated CCK-8 and gastrin-17, respectively. Immunization against desulfated CCK-8 had no effect on feed intake, ADG, carcass weight or carcass quality grade. Backfat thickness and carcass yield grade were reduced (P less than .05) by immunization. Organ weights at slaughter, including those of the pancreas and small intestines, were not affected by CCK-8 immunization, with the exception of the lungs, which were 16% lighter (P less than .01) in immunized lambs. In conclusion, active immunization against desulfated CCK-8 resulted in development of high antibody titers against desulfated and sulfated CCK-8. Immunization against CCK-8 decreased fat content of the carcass but failed to affect feed intake, carcass weight or ADG.

Quantitative restoration of advanced spermatogenic cells in adult male rats made azoospermic by active immunization against luteinizing hormone or gonadotropin-releasing hormone.

The ability of testosterone to quantitatively restore spermatogenesis in rats made azoospermic by active immunization against LH or GnRH was examined. Sexually mature adult male rats (n = 15/group) were actively immunized against ovine LH or GnRH-human serum globulin conjugate, while control rats (n = 10) were injected with saline. After 10 weeks of immunization, five rats per group were euthanized. For each rat, trunk blood was collected for determination of LH, FSH, and testosterone by RIA; seminiferous tubule fluid (STF) was collected from one testis per rat, and testosterone concentration was measured by RIA; the number of advanced spermatids per testis was determined from the contralateral testis. The results obtained after 10 weeks of treatment were as follows. 1) Serum LH and FSH were undetectable by RIA in GnRH-immunized rats. 2) Serum testosterone was undetectable in both the LH- and GnRH-immunized groups. 3) The testosterone concentration in STF (STF-T) was reduced from the control value of about 64 ng/ml to about 2 ng/ml in the LH- and GnRH-immunized rats. 4) LH- and GnRH-immunized rats were azoospermic. After the initial 10-week treatment period, five rats in each of the LH- and GnRH-immunized groups received 24-cm testosterone-filled polydimethylsiloxane (PDS-T) capsules (3 x 8 cm long) sc. The remaining immunized rats (n = 5/group) received empty capsules. Two months later, all rats were euthanized. Testis weights, serum testosterone, and STF-T concentrations remained significantly reduced in LH- and GnRH-immunized rats that did not receive testosterone supplementation, and the rats remained azoospermic. STF-T concentrations rose significantly (P less than 0.05) in the LH- and GnRH-immunized rats that received PDS-T, but were still significantly less (by approximately 80%) than the concentration in intact controls. Nonetheless, implantation of PDS-T caused restoration of advanced spermatogenic cells in the testes of both LH- and GnRH-immunized rats to numbers that were not significantly different from the number in controls. These data indicate that 1) testosterone is capable of quantitatively restoring spermatogenesis in rats actively immunized against LH or GnRH, suggesting that FSH may not be required for the restoration of spermatogenesis in adult rats; and 2) quantitatively complete restoration of spermatogenesis can occur at STF-T concentrations that are significantly reduced compared to those in intact controls.

Immunolocalization and concentrations of inhibin alpha in the ovine testis and excurrent duct system.

Inhibin was localized in the ovine testis, excurrent ducts, and accessory sex glands by using a rabbit antiserum against a synthetic polypeptide representing the first 30 amino acids of porcine inhibin alpha-subunit. Concentrations of inhibin in fluids entering and leaving the epididymis also were determined in a radioimmunoassay using the same antibody. In the testis, immunostaining of inhibin was conspicuous in the seminiferous epithelium. Leydig cells occasionally were stained and the tunica media of blood vessels always was stained. Intense staining was observed in the epithelia lining the rete testis and ductuli efferentes. Staining also was intense in the epithelium of the initial segment and proximal caput epididymidis, and became less intense along the length of the epididymis. These observations were consistent with concentrations of inhibin in rete testis fluid (8.2 pmol/ml) entering the ductuli efferentes and in cauda epididymal plasma (0.67 pmol/ml) leaving the epididymis. Epithelia of ampullary and vesicular glands and of some prostatic acini were positively stained, but bulbourethral glands were never stained. Adrenal cortex, some proximal convoluted tubules in the kidney, and transitional epithelium of the urethra also were stained. Based on radioimmunoassay data and fluid flow rates for the ram, it was concluded that almost all of the 328 pmol inhibin that enters the ductuli efferentes daily is endocytosed in the proximal parts of the excurrent duct system. The physiological role(s) for inhibin, or inhibin-like peptides, in the excurrent duct system remains speculative.

Pituitary and Leydig cell function in boars actively immunized against gonadotrophin-releasing hormone.

Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 micrograms GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 microgram/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0.05 and 0.2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P less than 0.05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.