Suproxide dismutase in human testis preparations

A protein fraction from human testis was structurally investigated. The main component of the fraction reported to contain inhibin-like activity was purified and analyzed by tryptic digestion. The peptides obtained identified the protein as an enzyme, superoxide dismutase, previously known to be present in seminal plasma. The results show that superoxide dismutase is a major enzyme, also of testicular material. They further demonstrate the importance of using pure fractions, and controls such as checks with structural analysis or synthetic peptides, in the work of elucidating the nature of inhibin and other hormonal peptides.     

Active immunization with a synthetic fragment of pig inhibin alpha-subunit increases ovulation rate and embryo production in superovulated ewes but season affects its efficiency.

Two experiments were designed to determine the effects of active immunization against one of two synthetic peptides from humans (inhibin-like peptide) or pigs (inhibin alpha-subunit) on antibody titres, ovulation rate and embryo production in ewes superovulated with 16 U ovine FSH. In Expt 1, during the breeding season, 30 ewes were subdivided into three groups: group I served as the non-immunized control; group II was immunized against inhibin-like peptide (100 micrograms inhibin-like peptide equivalent, followed by three booster injections); group III was immunized against pig inhibin alpha-subunit conjugated to human serum albumin (96 micrograms for the primary administration and 46 micrograms for the booster). In Expt 2, the efficiency of immunization against pig inhibin alpha-subunit on ovarian response and embryo production was evaluated during the non-breeding season in two groups of ewes (n = 12): group IV was a non-immunized control; Group V was immunized against pig inhibin alpha-subunit. During the breeding season, the ewes immunized against pig inhibin alpha-subunit showed higher antibody titres compared with the group immunized against inhibin-like peptide (P < 0.01) and a significant increase in ovulation rate (12.1) compared with both the control (5.0; P < 0.05) and the inhibin-like peptide-immunized group (3.1; P < 0.01). Immunization against pig inhibin alpha-subunit increased transferable embryo yield 4.5-fold (6.7 versus 1.5; P < 0.01) and improved embryo quality (94.6 versus 40.6%; P < 0.01). During the non-breeding season, immunization against pig inhibin alpha-subunit enhanced ovulation rate from 2.6 in the controls to 9.4 (P < 0.01) but did not affect transferable embryo production (3.9 versus 2.1; P > 0.05) and significantly lowered their quality (54.1 versus 100%; P < 0.01). In conclusion, active immunization against pig inhibin alpha-subunit can improve superovulatory response during the breeding season, while it appears to be unable to increase embryo yield during the seasonal anoestrus.     

De novo biosynthesis and localization of immunoreactive inhibin (10.7 kDa) in normal human stomach.

We have previously reported the occurrence of inhibin-like peptide in gastric juice of normal men. In the present investigation, normal gastric mucosa was shown to synthesize inhibin, in vitro, as measured by 3H-leucine incorporation (Maximum at 18 h). Furthermore, the immunohistochemical localization studies demonstrated its presence in the acid secreting parietal cells and basal region of foveolar epithelium of gastric mucosa. Surprisingly, the protein secreting zymogen cells remained unstained.     

A synthetic decapeptide analogue of human seminal plasma inhibin exhibiting specific suppression of follicle stimulating hormone release in rats

Synthesis and biological profile of a decapeptide analogue, [Tyr85, Cys(Acm)87]85-94 of human seminal plasma inhibin (HSPI) are described. The peptide suppressed the circulatory levels of follicle stimulating hormone (FSH) in adult male rats. No change in the levels of luteinizing hormone (LH) and prolactin (Prl) was observed. Whereas the peptide suppressed the release of both FSH and LH in vitro. This decapeptide is the smallest peptide reported so far to have FSH suppressing activity.     

Isolation of inhibin like peptides from human placenta

Two moieties of inhibin could be obtained by chromatography of partially purified preparations of inhibin from human placenta on Sephadex G-100, G-25 and ion exchange chromatography on diethylaminoethyl Sephadex A-50. The higher molecular weight moiety (14,000) designated as HPI-H appears to be similar to inhibin from human seminal plasma. While the lower molecular weight moiety (1500) designated as HPI-L appears to be similar to that of sheep testicular inhibin. The preparations from both human term placenta and human seminal plasma inhibited the binding of [¹²⁵I] human follicle stimulating hormone to rat testicular receptors. This effect of inhibins could be neutralized by antisera raised against corresponding polypeptide. Further these antibodies could neutralize endogenous inhibin resulting in 2 to 3 fold increase in serum follicle stimulating harmone levels, which could then be reversed by exogenous administration of the isolated inhibin preparations.     

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina

The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.     

Enhancement of in vitro release of FSH from rat pituitaries by a synthetic nonapeptide fragment of human seminal plasma inhibin

A synthetic C-terminal nonapeptide fragment of human seminal plasma inhibin preferentially enhances the basal release of FSH from rat pituitaries incubated in vitro, which indicates a direct action of the peptide on the pituitary. However, in the presence of LHRH, both FSH and LH release was increased particularly at higher doses of the nonapeptide. There was no change in prolactin release at 5 and 50 ng/ml but prolactin release was suppressed significantly at 500 ng/ml.     

Validation of coupled bioassay for inhibin by pituitary cell culture assay

Studies on the characterization of inhibin and inhibin-like factors have depended for the most part on the classicalin vitro pituitary cell culture assay. A major drawback with this assay is the turn-around time which is in the order of two weeks and consequently slows down purification efforts. The 24 h bioassay for inhibin has been found to be sufficiently sensitive and also statistically valid. Unfortunately, based as it is on a secondary response, ambiguities arise in interpreting the results. By including a parallel assay in which the mice are primed with human menopausal gonadotropin rather than human chorionic gonadotropin, it was possible to device the coupled bioassay. This enables distinguishing inhibin-like factors acting to suppress pituitary follicle stimulating hormone output from those acting at the level of gonads. In this study the coupled assay for inhibin has been compared with the classical pituitary cell culture assay in order to assess its biological and statistical validity. The data validates the bioassay on both the above counts and when considered in conjunction with the short turn-around time suggests that this assay can be highly useful in studies on isolation of inhibin from various sources.     

Endometrium--an extragonadal source of inhibin.

Using polyclonal antibodies generated against human seminal plasma inhibin (10.5 KDa), immunocytochemical localization was carried out in paraffin embedded tissue sections of human endometrial biopsies obtained at various phases of the menstrual cycle. A positive reaction which indicated the presence of inhibin was characterized by the presence of golden yellow or brown colour in the cytoplasm of epithelial cells that formed the glands as well as the luminal lining. The stromal cells however, showed negative staining. In early proliferative phase, the endometrial glands exhibited weak positive staining for inhibin which gradually increased and was intense in late follicular and early secretory phases. The intensity of the staining although was not diminished in the glandular epithelium in the mid as well as late secretory phases, the number of cells showing positive staining appeared to be reduced. Incubation of endometrial biopsies in vitro with labelled amino acid and immunoprecipitation of newly synthesized protein with specific antibodies to inhibin indicated that endometrium is capable of de novo synthesizing inhibin. The above results suggest that endometrium is an extra ovarian source of inhibin and the possible role of endometrial peptide in sperm fertilizing capabilities as well as in pre and post implantation events is suggested.     

Seasonal changes in inhibin activity in seminal plasma and serum concentrations of FSH, LH and testosterone in the male goat (Capra hircus)

Inhibin activity in goat seminal plasma was measured by in vitro assay throughout successive 9 months and its relationship with the serum FSH, LH and testosterone concentrations was investigated. Total inhibin activity (TIA) in seminal plasma gradually increased from spring to summer, reduced in autumn (P<0.05) and recovered toward winter (P<0.05). Serum FSH and LH reached a peak in mid-summer (P<0.01) and returned to the low levels in autumn. Serum testosterone also increased in mid-summer and kept the high levels until the early winter (P<0.05). Some positive correlation was found in monthly levels between seminal TIA and serum FSH (r=0.305; P<0.05). Results suggest that the summer increase of inhibin activity in seminal plasma relates with the mid-summer rise of serum FSH levels in the male goat.     

Rat ventral prostate as an independent source of inhibin-like material

Castration had no effect on prostatic inhibin-like activity as compared to normal controls. Consequent upon castration there is a significant reduction in the inhibin content of the ventral prostate on a per gland basis. However, when expressed as a function of protein content, there is no change. The data suggest that the very same epithelial cells which under the influence of androgen carry out the characteristic exocrine secretory activity are responsible for the synthesis and secretion of inhibin-like material.     

In vivo suppression of pituitary and circulatory follicle stimulating hormone by human seminal plasma inhibin

Human seminal plasma inhibin (HSPI), of prostatic origin, has been shown to bring about a dose dependent suppression in pituitary and circulatory FSH concentrations in intact rats. No significant changes in LH levels either in pituitary or in circulation were observed at the doses used. This has further been substantiated by an immunocytochemical staining. A marked reduction in staining intensity for FSH was observed in the pituitary of inhibin treated rats as compared to the controls. None of the purified inhibin peptides from other sources have so far been reported to act on pituitary FSH in vivo. This study thus, for the first time demonstrates an in vivo effect of inhibin (HSPI) on pituitary FSH concentration and secretion.     

Complete amino acid sequence of human seminal plasma beta-inhibin. Prediction of post Gln-Arg cleavage as a maturation site

The complete sequence of a 94 amino acid human seminal plasma polypeptide exhibiting inhibin-like activity is presented. This molecule, called beta-inhibin, selectively and specifically suppresses the release of pituitary FSH in vivo as well as in vitro. It does not affect the secretion of LH. Such a novel acidic protein contains a very basic C-terminal segment which is easily cleaved by mild tryptic digestion. It is predicted that the FSH inhibiting activity may reside within this region of the molecule. This would imply a post Gln-Arg cleavage to release the basic C-terminal active moiety.     

Peptides of postulated inhibin activity. Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment

A 94-residue polypeptide isolated from human seminal plasma and its chemically synthesized C-terminal 28-residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH-stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94-residue peptide and the synthetic replicate of its C-terminal 28-residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.     

Does inhibin have intrinsic prolactin suppressing activity?

Evidence to demonstrate suppressive effect of inhibin on prolactin has been presented. The inhibin preparations derived from human testicular tissue, human seminal plasma and porcine follicular fluid were tested and all the three preparations were found to exhibit prolactin suppressing activity.     

Gonadotrophin-inhibiting activity in seminal plasma from intact and vasectomized bulls

Bull seminal plasma administered to male rats at the time of castration inhibited the rise in the levels of FSH and LH otherwise seen in the serum 24 h later. That the gonadotrophin-inhibiting activity was also present in the seminal plasma from vasectomized bulls suggests that it was not of testicular origin. Although the substance with gonadotrophin-inhibiting activity was a protein, it may be chemically distinct from inhibin.     

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.     

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.