In order to study if the reduction of serum T, although mild, might modify the qualitative protein composition of spermatozoa, we selected five patients affected by hypotestosteronemia due to isolated LH deficiency. However, there is a minimally required T concentration below which spermatogenesis is affected, leading to male infertility (23). The analysis of seminal parameters in those patients confirmed that spermatogenesis was conserved because of FSH levels, since sperm could be observed in the ejaculate. The relative quantification of proteins was achieved by dividing the intensity of reporter ions of HCD MS2 spectra for secondary hypogonadism patients pool (TMT-127) with the controls pool (TMT-126), which were obtained using Proteome Discoverer software. Human semen samples were obtained from 6 male patients diagnosed with secondary hypogonadism due to LH deficiency and 5 fertile controls at the andrological clinic of Fondazione Policlinico "A. Six male hypogonadic patients aged between 25- and 55-year-old with secondary hypogonadism due to isolated LH deficiency were consecutively enrolled for this study.
This dysfunction not only affects the quantity of sperm but also compromises their structural integrity and ability to fertilize an egg. They create a blood testis barrier, provide nutrients, and remove waste from developing sperm cells. Sertoli cells, often called the "nurse cells" of the testes, play a vital role in sperm development. Spermatogenesis is the process through which immature germ cells in the testes mature into functional sperm. LH travels through the bloodstream to the testes, where it stimulates the Leydig cells to produce testosterone.
Likewise, to assess sample RNA integrity, protamine 1 (PRM1) was targeted as a positive control of a sperm-specific intact RNA (Supplementary Table 1). For Diff-Quick staining, sperm smears were prepared by placing 5 µl of the sample onto a slide and pulling it out into a smear using a second slide followed by air drying for 20–30 s. The recovered sperm cells were then resuspended in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) and subjected to a residual leukocyte depletion using Dynabeads™ CD45 (Invitrogen, Carlsbad, CA, USA). Briefly, the sperm samples were centrifuged through a 50% Percoll gradient at 400 g for 30 min at room temperature (RT) without brake. The ejaculates were washed with PBS and the sperm cells were selected after 50% Percoll™ gradient (GE Healthcare, Uppsala, Sweden), as previously described (17, 18). Statistical analysis has been carried out with SPSS v18.0 (IBM Corp., Armonk, NY, USA). The reference values of the studied hormones are reported in Table 1.
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Gemelli" IRCCS in Rome (Italy), for the determination of T, estradiol (E2), sex hormone–binding globulin (SHBG), LH, and FSH. A pituitary magnetic resonance imaging (MRI) was performed in all patients to confirm the diagnosis of pituitary disease. FSH is particularly important for establishing a normal and functional Sertoli cell population, LH for promoting the production of T, whereas androgen action is needed for the fulfillment of germ cell development. However, isolated LH deficiency is a rare clinical condition, originating from a dysregulation in the hypothalamus-pituitary GnRH-LH-testosterone axis, which results in an isolated dysregulation of LH secretion, with a normal or low-normal FSH secretion (5). Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer.