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P Berger

Publications and source records attributed to P Berger.

At least 109 records · Page 6Linked to original sources

Prolactin gene expression and prolactin protein in premenopausal and postmenopausal human ovaries.

OBJECTIVE: To investigate intraovarian prolactin and prolactin-receptor gene expression and to assess local prolactin synthesis with emphasis on possible differences between premenopausal and postmenopausal status. DESIGN: The RNA extracted from human premenopausal and postmenopausal tissues was subjected to reverse transcription and polymerase chain reaction by using prolactin-specific intron- and exon-spanning primers. Prolactin-receptor expression was investigated accordingly. The amplified complementary DNA fragments were analyzed by gel electrophoresis and restriction enzyme mapping. Local prolactin hormone synthesis was verified by a time-resolved immunofluorometric assay based on our monoclonal antibodies. RESULT(S): Prolactin and prolactin-receptor gene expression was observed in all analyzed human ovaries (n = 18). Several other human tissue specimens, such as lung and kidney, served as negative control tissues. Significantly elevated concentrations of prolactin were detected in cytosolic extracts of premenopausal (n = 6; mean +/- SD; 20.6 +/- 3.3 ng/g tissue wet weight) versus postmenopausal (n = 6; 3.6 +/- 3.0 ng/g tissue wet weight) ovaries. CONCLUSION(S): The human ovary not only serves as a target for endocrine prolactin action but also as a site of local prolactin hormone production. In agreement with previous reports on extrapituitary sources of prolactin, we consider prolactin as a hormone as well as an autocrine or paracrine growth or regulatory factor. Significantly increased concentrations of prolactin in premenopausal ovarian tissue verifies its role in human reproduction.

Adult↗

Tumor markers in hydrocele fluids of patients with benign and malignant scrotal diseases.

PURPOSE: We evaluated the presence of human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP) and a panel of other tumor markers in the compartment next to the tumor (that is, the malignant hydrocele fluid). MATERIALS AND METHODS: We measured hCG, AFP, neuron-specific enolase, carcinoembryonic antigen and cytokeratin-19 fragments in cubital vein sera and in hydrocele fluids of patients with testicular cancer. Results were compared with those obtained from hydrocele fluids of patients with benign disease. RESULTS: All tumor markers remained under the respective cutoff values in benign hydroceles. In patients with pure seminomas, hCG levels were elevated in 66% of hydroceles but only once in peripheral sera, whereas AFP remained low in both compartments. Furthermore, of 11 cases of nonseminomatous germ cell tumor hydrocele fluids, 3 with negative peripheral tumor marker values had to be reclassified marker positive, of which 2 showed elevated hCG levels and 1 had increased levels of AFP. Significant changes of neuron-specific enolase and carcinoembryonic antigen concentrations could not be observed. However, a cytokeratin-19 fragment measured by Cyfra 21-1 assay was elevated in 2 of 3 seminomatous and in 4 of 8 nonseminomatous hydroceles. CONCLUSIONS: These data give a new insight into the in vivo secretion pattern of testicular germ cell neoplasms, which demonstrates that the term "marker negative" should be restricted to selected cases of testicular cancer. Analysis of tumor markers in hydrocele fluids may be a helpful tool in patients with scrotal swelling if clinical and sonographic results remain uncertain.

Adolescent↗

Quadruple immunofluorescence: a direct visualization method.

We describe fluorescence immunostaining of four different antigens in the same section. The fluorochrome conjugates used show highest emission in the blue, green, yellow, and red regions of the visible light spectrum, respectively. Specially designed single fluorochrome filter combinations allow selective visualization of each fluorochrome label in turn, without visible crosstalk with the others.

Adrenocorticotropic Hormone↗

Selective growth hormone/placental lactogen gene transcription and hormone production in pre- and postmenopausal human ovaries.

In addition to effects of pituitary-derived gonadotropins, human GH modulates and regulates intraovarian reproductive processes in a dose-dependent manner via the endocrine GHRH/GH/insulin-like-growth-factor I (IGF-I) axis. Based on increasing evidence that ovarian regulation involves a complex system of putative para/autocrine factors, we investigated the possibility of gene-selective intraovarian GH/placental lactogen (PL) hormone production, with emphasis on differences between pre- and postmenopause. Analysis of both premenopausal (n = 8) and postmenopausal (n = 10) ovarian-derived messenger ribonucleic acid by reverse transcription-PCR, which amplifies all major gene products of the five-member GH/PL gene cluster GH-N, GH-V, PL-A/B, and PL-L, revealed specific transcripts in all specimens. Their share in gene selective expression by analytical restriction enzyme digestion was determined. The expression pattern of GH/PL messenger ribonucleic acid shows PL-A/B > GH-N, which sets it apart from those of pituitary and placenta. Local production of the respective protein hormones was verified by two time-resolved immunofluorometric assays for human PL-A/B and GH-N; significant amounts of these hormones were detected in cytosolic extracts of premenopausal (n = 6; 555.5 +/- 171 ng PL-A/B and 0.8 +/- 0.6 ng GH-N/g tissue wet wt) and postmenopausal (n = 6; 5.2 +/- 2.7 ng PL-A/B and 0.9 +/- 0.6 ng GH-N/g tissue wet wt) ovaries. No difference was observed between pre- and postmenopausal ovarian GH-N contents, but PL values were 2-3 orders of magnitude lower in postmenopausal tissue (P < 0.001). Serum levels of healthy premenopausal (n = 21) and postmenopausal (n = 16) women were less than 0.02 ng PL/mL. In summary, ovarian-derived GH-N and PL-A/B synthesis correlates well with the established local cascade of GHRH, GHRH receptor, GH receptor, IGF-I, and IGF-I receptor as a putative para/autocrine regulator of ovarian reproductive function.

Adult↗

[The first panic attack: a missed chance for prevention of panic disorder and its complications].

OBJECTIVE: Assessment and analysis of first medical consultations and their significance in panic disorder patients. METHODS: 90 panic disorder patients were interviewed concerning their experiences with the medical system at the time of their first panic attack. RESULTS: Panic disorder patients contacted mostly non-psychiatric medical services at the time of their first panic attack. The correct diagnosis was established in only 4 cases (5.6%). CONCLUSIONS: An important chance for secondary prevention of the development of panic disorder is missed, probably due to the poor education of physicians on panic attacks.

Adolescent↗

Immunochemical mapping of gonadotropins.

As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.

Amino Acid Sequence↗

High frequency of EEG and MRI brain abnormalities in panic disorder.

The frequency and quality of brain abnormalities in panic disorder (PD) were assessed with magnetic resonance imaging (MRI). The use of electroencephalography (EEG) to detect PD patients with a high probability of morphologic brain abnormalities was also explored. Consecutive PD patients (n = 120) were screened with routine EEG examinations and were divided into the following subgroups on the basis of their EEG findings: patients with non-epileptic EEG abnormalities (EEG-A group, n = 28), matched patients with normal EEG results (EEG-N group, n = 28) and matched healthy controls (n = 28). PD patients showed a higher than expected rate of non-epileptic EEG abnormalities (29.2%; 35 of 120). EEG screening was effective in identifying patients with a high probability of morphologic brain abnormalities. MRI abnormalities were found in 60.7% of the EEG-A patients, 17.9% of the EEG-N patients, and only 3.6% of the controls. A high frequency of septo-hippocampal abnormalities was found. Further research should focus on attempts to subtype PD on the basis of neuroanatomic and functional brain abnormalities.

Adult↗

Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.

The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.

Animals↗

Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model.

We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.

Antibodies, Monoclonal↗

Identification and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit.

The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.

Chorionic Gonadotropin, beta Subunit, Human↗

Enhancement in antigen binding by a combination of synergy and antibody capture.

The effects of orientating pairs of synergistic monoclonal antibodies (mAb) on binding of human chorionic gonadotropin (hCG) was studied by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Antibody synergy towards hCG required two functionally intact antibodies located adjacent to each other and with different epitope specificities. We investigated whether immobilization procedures avoiding protein denaturation, increasing proper orientation and promoting higher molecular flexibility of the synergistic mAb resulted in significantly enhanced antigen, binding. Synergistic mAb pairs captured through their Fc-region by protein G or a polyclonal serum against the Fc-part of mouse IgG could be used at 10-fold lower coating concentrations to achieve maximal binding of the analyte as compared with the same mAb pairs coated directly onto polystyrene. The synergistic effect observed with protein A used as capture varied greatly with the subclasses of the two synergistic antibodies employed. Scatchard analysis revealed that the number of functionally synergistic antibody sites participating in the binding of hCG for one mAb pair was about 10 times higher for the protein G-captured as compared with the directly coated synergistic pair. Biotinylated synergistic mAb pairs, coated directly or captured by streptavidin, did not display any enhanced antigen binding when tested in SPR or ELISA. With SPR, synergy was only observed when the synergistic mAb had been captured through their Fc-region. Using protein G or a polyclonal rabbit anti-IgG1 serum as capture reagents in SPR, synergistic triple mAb combinations against hCG were demonstrated.

Animals↗

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion.

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV-1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin-1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.

Amino Acid Sequence↗

Expression of the human chorionic gonadotropin-beta gene cluster in human pituitaries and alternate use of exon 1.

Previous studies have indicated that in addition to other glycoprotein hormones, the pituitary gland produces small amounts of hCG beta, the classical pregnancy and tumor marker. At the gene transcription level, definitive proof for hCG beta messenger ribonucleic acid transcription was still lacking, largely due to the 90% homology to hLH beta at the DNA sequence level, which renders specific hCG detection in the presence of a vast excess of LH difficult. We investigated both the presence of hCG beta messenger ribonucleic acid and the protein itself in normal human female postmenopausal (n = 4) and male pituitaries (n = 2). Reverse transcription-PCR and subsequent restriction enzyme analysis revealed that the hCG beta 3, 5, 7, and 8 genes coding for genuine hCG beta were transcribed in all pituitaries. Additionally, three alternatively spliced gene products derived from hCG beta genes 1 and 2 were detected and verified by single strand sequencing of the complementary DNAs. The most abundant fragment (244 bp) showed a point mutation (T-->A) in the splice donor site for the first intron, resulting in an alternate use of exon 1 and a frame shift in the open reading frame that might give rise to a hypothetical protein, 132 amino acids in length. With regard to protein synthesis, we confirmed the pituitary as the site of production for hCG beta by reverse phase high performance liquid chromatography and subsequent immunoradiometric assays, including a monoclonal antibody directed against the unique C-terminal extension of hCG beta.

Aged↗

An effectiveness community-based clinical trial of Respirgard II and Fisoneb nebulizers for Pneumocystis carinii prophylaxis with aerosol pentamidine in HIV-infected individuals. Toronto Aerosol Pentamidine Study (TAPS) Group.

STUDY OBJECTIVE: To compare the effectiveness of a standard jet nebulizer, Respirgard II, and a standard ultrasonic nebulizer, Fisoneb, for the administration of aerosolized pentamidine (AP) as primary and secondary prophylaxis against Pneumocystis carinii pneumonia (PCP) in HIV-infected individuals. DESIGN: A retrospective, nonrandomized, parallel group comparative study. SETTING: Patients were enrolled in a community-based AP program (APP) between May 1989 and April 1992 in Ontario, Canada. They received AP in either (1) a centralized treatment facility ("clinic") or (2) their attending physician's office or regionalized centers ("nonclinic"). Clinic administration of pentamidine was via Fisomeb; nonclinic via Respirgard II. PATIENTS: The study group comprised of 1,762 HIV-infected individuals requiring AP for either primary (CD4 < 200/mm3) or secondary PCP prophylaxis. Of these, 1,151 used Fisoneb (clinic) and 611 used Respirgard II (nonclinic). RESULTS: In the primary prophylaxis group, 41 of the 892 patients using Fisoneb (4.6%; mean follow-up, 18 months) compared with 16 of 435 patients using Respirgard II (3.7%; mean follow-up, 14.6 months) developed PCP (p = 0.44). A total of 28 of 259 (10.8%; mean follow-up, 15.3 months) patients using Fisoneb for secondary prophylaxis compared with 11 of 176 (6.3%; mean follow-up, 14.4 months) patients using Respirgard II for secondary prophylaxis developed PCP (p = 0.1). CONCLUSIONS: Despite the difference in dosage (120 mg/mo vs 300 mg/mo), type of nebulizer (ultrasonic vs jet), and frequency of administration (twice vs once monthly), the results of this study indicate that both regimens of AP provide comparable protection against PCP. This study further supports the effectiveness of AP as a solid second-line prophylaxis for HIV-infected individuals who are intolerant to trimethoprim/sulfamethoxazole or dapsone.

AIDS-Related Opportunistic Infections↗

Long-term results of patients with clinical stage C prostate cancer treated by phototherapy and early orchiectomy. A retrospective analysis of 169 cases.

BACKGROUND: To evaluate the value of radiotherapy and immediate hormonal therapy in the treatment of stage C prostate cancer. PATIENTS AND METHOD: From 1977 to 1986, 169 patients with clinically stage C prostate cancer underwent irradiation with curative intent following early orchiectomy. Sixty-four patients had a transurethral resection, 22 patients a prostatectomy and 83 patients had only a biopsy. In 38 patients a grade Ia/b tumor was found, in 78 patients a grade IIa/b tumor and in 43 patients a grade IIIa/b tumor using the German grade of malignancy. Treatment fields included the prostate, the seminal vesicles and the locoregional lymphatics. Until 1979 the dose was 60 Gy for the tumor encompassing isodose and from then on 65 Gy with a single dose of 2 Gy. RESULTS: With a median follow-up of 98 months, the overall survival rate for 8 and 10 years was 51% and 37% and the cause specific survival rate was 84% and 77%, respectively. Thirty-two patients (19%) developed distant metastases. Patients with local tumor control (n = 148) had a significantly better overall survival rate of 45% for 10 years compared to patients with clinical local progression of disease (n = 21) of 22% (p < 0.05). Multivariate analysis showed the grade of malignancy and local control as independent factors for overall survival and cause-specific survival (p < 0.05). Twenty-three patients (14%) had at least one late side effect for the rectum or the bladder, in almost all cases grade I or II. Five patients (3%) showed severe late side effects RTOG grade III (n = 2) or IV (n = 3). One patient had a colostomy, in 2 patients a severe haemorrhagic cystitis was seen. CONCLUSIONS: Radiotherapy with photons and early orchiectomy for patients with stage C prostate cancer achieves high local control rates and a 30% to 40% 10-year survival rate with a low incidence of late side effects. The value of the radiotherapy of the locoregional lymphatics remains controversial.

Adult↗

[The testis as eutopic production site of human growth hormone, placental lactogen and prolactin: possible autocrine/paracrine effects on testicular function].

The human (h) protein hormones, growth hormone (hGH-N) and prolactin (hPRL), are mainly produced in the pituitary, whereas the human placenta expresses the other four members of the protein hormone gene family, designated placental lactogens (PL-A, PL-B, PL-L) and growth hormone variant (GH-V), GH-N stimulates somatic growth, supports nitrogen-, phosphate- and potassium retention and promotes lipolytic and anabolic metabolism, whereas PRL acts on the mammary gland and induces mammogenesis, lactogenesis and galactopoesis. Both hyperprolactinemia and growth hormone deficiency affect the onset of puberty and reproduction in man and mice. In addition to the glycoprotein hormones, these hormones play a role in the maintenance of testicular function. Our group previously demonstrated eutopic production of glycoprotein hormones hLH (human luteinizing hormone) and hCG (human chorionic gonadotropin) in the testis. We have now extended our investigations to the local testicular expression of protein hormones. By means of the molecular biology techniques of the reverse transcription-polymerase chain reaction (RT-PCR), Southern blot and by restriction endonuclease analyses of the generated PCR products we demonstrated the eutopic expression of genes coding for the protein hormones. GH-N gene transcripts were detected only in the pituitary and abundant PL-A/B and a few GH-V gene transcripts were demonstrable in the placenta. In contrast, in the testis GH/PL and PRL genes are transcribed. Since testicular protein hormone gene expression is rather low, these hormones may act locally and not as systemic factors; they presumably modulate the LH/CG-mediated testosterone biosynthesis and/or may act on the spermatogenesis.

Animals↗