Reproductive Biology Laboratory Research Articles

The revision of the Bioethics Act, two years on: major principles and consequences for practice in the reproductive biology laboratory

Since 1994, in France, bioethics law has set the regulatory framework for Medically Assisted Reproduction (MAR). The latest revision of the law of August 2, 2021, is characterized by major upheavals in the field of MAR and intervenes in several areas: the purpose and conditions to access to MAR, access to origins in the case of gamete or embryo donation, and gametes cryopreservation without medical indication. Indeed, the law authorizes, because of a strong societal demand, the extension of sperm donation to couples of women and unmarried women, as well as the possibility for any person to preserve his/her gametes if he/she meets the age criteria defined by decree. Finally, the law opens the possibility for people born following gamete or embryo donation to have access, from their 18th anniversary, to identifying and/or non-identifying data. These new measures have led to a very important number of MAR requests to fertility and donation centers, and have required the implementation of new circuits in order to harmonize care, without discrimination or prioritization.

Array-based DNA methylation profiling reveals peripheral blood differential methylation in male infertility

To study peripheral blood DNA differential methylation in oligozoospermic infertile men in comparison with normozoospermic fertile controls. Case-control study. Reproductive biology laboratory. Azoospermic and oligozoospermic infertile patients (n = 6) and normozoospermic fertile controls (n = 6) in the discovery phase, and oligo/asthenozoospermic infertile men (n = 11) and normozoospermic fertile controls (n = 10) in the validation phase. Blood samples drawn from all participants, DNA isolation and methylation analysis. DNA methylation values analyzed using genomewide methylation 450K BeadChip array, followed by deep sequencing of selected regions for methylation analysis in the neighborhood regions of differentially methylated CpGs. We found 329 differentially methylated CpG spots, out of which 245 referred to the genes, representing 170 genes. Deep-sequencing analysis confirmed the methylation pattern suggested by 450K array. A thorough literature search suggested that 38 genes play roles in spermatogenesis (PDHA2, PARP12, FHIT, RPTOR, GSTM1, GSTM5, MAGI2, BCAN, DDB2, KDM4C, AGPAT3, CAMTA1, CCR6, CUX1, DNAH17, ELMO1, FNDC3B, GNRHR, HDAC4, IRS2, LIF, SMAD3, SOD3, TALDO1, TRIM27, GAA, PAX8, RNF39, HLA-C, HLA-DRB6), are testis enriched (NFATC1, NMNAT3, PIAS2, SRPK2, WDR36, WWP2), or show methylation differences between infertile cases and controls (PTPRN2, RPH3AL). We found a statistically significant correlation between peripheral blood DNA methylation and male infertility, raising the hope that epigenome-based blood markers can be used for screening male infertility risk. The study also identified new candidates for spermatogenesis and fertility.

Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) invitro. Experimental basic science study. Reproductive biology laboratory. ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. Invitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70by means of immunofluorescent assay. The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation invitro. The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.

Advanced ultrasound imaging: Insights from perfusion and molecular imaging

Advanced ultrasound imaging: Insights from perfusion and molecular imaging

Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

To study the ability of human spermatogonial stem cells (hSSCs) to proliferate invitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create invitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA-)/epithelial cell surface antigen (EPCAM+) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells invitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA-/EPCAM+ resulted in an enrichment of 27% VASA+/UTF1+ hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm2 after 2weeks invitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4weeks invitro. We found that hSSCs show limited proliferation invitro under mSSC culture conditions. Coculture of HLA-/EPCAM+ sorted cells with testicular feeders improved the germ cell/somatic cell ratio.

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of testicular cells from fresh and frozen tissues during long-term culture. Human testicular cells derived from fresh (n = 3) and cryopreserved (n = 3) tissues were cultured for 2 months and analyzed with quantitative reverse-transcription polymerase chain reaction and immunofluorescence. Spermatogonia including spermatogonial stem cells (SSCs) were reliably detected by combining VASA, a germ cell marker, with UCHL1, a marker expressed by spermatogonia. The established markers STAR, ACTA2, and SOX9 were used to analyze the presence of Leydig cells, peritubular myoid cells, and Sertoli cells, respectively. No obvious differences were found between the cultures initiated from fresh or cryopreserved tissues. Single or small groups of SSCs (VASA(+)/UCHL1(+)) were detected in considerable amounts up to 1 month of culture, but infrequently after 2 months. SSCs were found attached to the feeder monolayer, which expressed markers for Sertoli cells, Leydig cells, and peritubular myoid cells. In addition, VASA(-)/UCHL1(+) cells, most likely originating from the interstitium, also contributed to this monolayer. Apart from Sertoli cells, all somatic cell types could be detected throughout the culture period. Testicular tissue can be cryopreserved before long-term culture without modifying its outcome, which encourages implementation of testicular tissue banking for fertility preservation. However, because of the limited numbers of SSCs available after 2 months, further exploration and optimization of the culture system is needed.

Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues

To compare mechanical dissociation, employing the Medimachine system, and enzymatic digestion of human testicular tissues with respect to the proportion of spermatogonia and somatic cells, with the long-term objective of establishing human spermatogonial cultures. Experimental basic science study. Reproductive biology laboratory. Testicular tissues were obtained from patients with gender dysphoria on the day of sex reassignment surgery. On the basis of the histological evaluation, tissue samples with complete spermatogenesis (fresh, n = 6; cryopreserved, n = 7) and with meiotic arrest (cryopreserved, n = 4) were selected. None. The composition of testicular cell suspensions was assessed performing quantitative real-time polymerase chain reaction (qPCR) analyses for germ cell-specific (FGFR3, SALL4, UTF1, MAGE-A4) and somatic marker genes (ACTA2 and VIM). Additionally, flow-cytometric analyses were used to evaluate the percentage of SALL4-and vimentin-positive cells. While Medimachine dissociation yielded higher cell numbers in all patient groups, viability of cells was highly variable and correlated with the histological status of the tissue. Interestingly, qPCR analysis revealed a significantly decreased expression of the somatic marker genes ACTA2 and VIM and an increased expression of the spermatogonial marker genes FGFR3 and SALL4 after Medimachine dissociation. These findings were corroborated by flow-cytometric analyses that demonstrated that the proportion of SALL4-positive cells was up to 4 times higher after mechanical dissociation. Medimachine dissociation of human testicular tissues is comparably fast and leads to an enrichment of SALL4-positive spermatogonia. The use of this method may therefore constitute an advantage for the establishment of human spermatogonial cell cultures.

Human chorionic gonadotropin stimulates spheroid attachment on fallopian tube epithelial cells through the mitogen-activated protein kinase pathway and down-regulation of olfactomedin-1

To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro. Experimental study. Reproductive biology laboratory. Healthy nonpregnant women. No patient interventions. Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay. Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression. Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression.

Genetic and epigenetic stability of human spermatogonial stem cells during long-term culture

To determine the genetic and epigenetic stability of human spermatogonial stem cells (SSCs) during long-term culture. Experimental basic science study. Reproductive biology laboratory. Cryopreserved human testicular tissue from two prostate cancer patients with normal spermatogenesis. None. Testicular cells before and 50days after culturing were subjected to ITGA6 magnetic-activated cell sorting to enrich for SSCs. Individual spermatogonia were analyzed for aneuploidies with the use of single-cell 24-chromosome screening. Furthermore, the DNA methylation statuses of the paternally imprinted genes H19, H19-DMR (differentially methylated region), and MEG3 and the maternally imprinted genes KCNQ1OT1 and PEG3 were identified by means of bisulfite sequencing. Aneuploidy screening showed euploidy with no chromosomal abnormalities in all cultured and most noncultured spermatogonia from both patients. The methylation assays demonstrated demethylation of the paternally imprinted genes H19, H19-DMR, and MEG3 of 11%-28%, 43%-68%, and 18%-26%, respectively, and increased methylation of the maternally imprinted genes PEG 3 and KCNQ1OT of 13%-50% and 30%-38%, respectively, during culture. In the current culture system for human SSCs propagation, genomic stability is preserved, which is important for future clinical use. Whether the observed changes in methylation status have consequences on functionality of SSCs or health of offspring derived from transplanted SSCs requires further investigation.

Enrichment of spermatogonial stem cells from long-term cultured human testicular cells

To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6+, HLA-/ITGA6+, GPR125+, and HLA-/GPR125+ magnetic-assisted cell sorting (MACS). Experimental basic science study. Reproductive biology laboratory. Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. Compared with the nonsorted cultured testicular cells, only the ITGA6+ and HLA-/GPR125+ sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6+. Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings.

Limiting donor conceptions to six: Time for change

In terms of the Regulations Relating to the Artificial Fertilisation of Persons (R.175) of the National Health Act, when gametes are removed or withdrawn from the body of a gamete donor, this is done for the purpose of artificial fertilisation. In the Regulations, artificial fertilisation is defined as '... the introduction by other than natural means of a male gamete or gametes into the internal reproductive organs of a female person for the purpose of human reproduction ...'. All information regarding the donated gametes is to be stored in an electronic central databank. No further gametes can be removed or withdrawn from the body of a donor once six children have been conceived through artificial fertilisation using the gametes of that donor. If a competent person involved in this aspect of reproductive medicine is informed or suspects that the maximum number of conceptions have occurred, s/he must make the donor aware of this and in addition must inform the donor that s/he may not make any further donation of gametes. This information must also be relayed to the central databank. A competent person is defined very broadly as a medical practitioner specialising in gynaecology with training in reproductive medicine, or a medical scientist, medical technologist or clinical technologist with training in reproductive biology and related laboratory procedures.

Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study

To study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system. Experimental laboratory study. Reproductive biology laboratory, academic medical center. Acute lymphoblastic leukemia cells from three patients and testicular cells from three other patients. Acute lymphoblastic leukemia cells were cultured alone or in combination with testicular cells, at various concentrations, in a system that has recently been developed to propagate human spermatogonial stem cells. Viability of ALL and testicular cells during culture was evaluated by flow cytometry using markers for live/dead cells. Furthermore, the presence of ALL cells among testicular cells was determined by highly sensitive (1:10,000 to 1:100,000 cells) patient-specific antigen-receptor minimal residual disease polymerase chain reaction. The presence of spermatogonia at the end of culture was determined by reverse transcription-polymerase chain reaction for ZBTB16, UCHL1, and GPR125. The ALL cells cultured separately did not survive beyond 14 days of culture. When cultured together with testicular cells, even at extremely high initial concentrations (40% ALL cells), ALL cells were undetectable beyond 26 days of culture. Reverse transcription-polymerase chain reaction confirmed the presence of spermatogonia at the end of the culture period. Our pilot study shows that the described testicular cell culture system not only allows for efficient propagation of spermatogonial stem cells but also eliminates contaminating ALL cells.

Testicular Perfusion Apparatus

A reproductive biology laboratory requires to provide reliable instruments for regular use of research workers. To understand an organ and perform scientific study and reach on a conclusion good instruments and techniques are essential. Such a suitable one is not available in case of testicular study. The present study is aimed to clear this lacuna. A new testicular perfusion apparatus for rabbit and rat is designed and fabricated with glass. The glass blowing instrument available is used. The perfusion fluid preferred is Krebs Ringer bicarbonate buffer (pH 7.4). Quartz wool provided in stem tube filters emboli present in it. Perfusate temperature is maintained to that of testis. A 26-gauge needle is introduced into the spermatic artery to allow entry of perfusate into testis. Method for the collection of perfusate leaving testis is available. This permits to measure the level of an injected substance before its entry through perfusion fluid into the testis, which shall be compared in the fluid after completion of perfusion. Initially few animal testes were used and found the instrument to be functioning well. A new simple perfusion apparatus for testes of rabbit and rat is designed. The apparatus made out of glass could be prepared with the help of glass blowing facility and used regularly in any laboratory.

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium

To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. A reproductive biology laboratory. Not applicable. Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM). Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.

Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME)

With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (μm(2))/head area (μm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.

Bilan d’une azoospermie et évaluation histologique de la spermatogenèse

Azoospermia may be obstructive (blockage of the genital ducts) or non-obstructive (a lack of testicular production). The distinction is based on an ensemble of clinical, spermiological, hormonal, ultrasound, genetic and histological data. Azoospermia is the main indication for testicular biopsy for therapeutic and diagnostic purposes. Testicular spermatozoids are processed in the reproductive biology laboratory (simultaneously with oocyte retrieval or not) for in vitro fertilization with intra-cytoplasmic sperm injection. The histological study of spermatogenesis is usually performed on a testicular biopsy sample taken at the same time and provides additional diagnostic information on infertility. Histological alterations in the testicular tissues are frequently observed in azoospermic men. In non-obstructive azoospermia, three histological situations prevail: hypospermatogenesis, Sertoli-cell-only syndrome and germ cell arrest. One can distinguish between pure forms (in which all the seminiferous tubules have the same appearance) and mixed forms (in which the tubules' aspects are heterogeneous). Hypospermatogenesis is highly prevalent in azoospermia and is characterized by a low, basal level of spermatozoid production. The prevalence of Sertoli-cell-only syndrome varies from 27 to 68% and the mean spermatozoid recovery rate is between 16 and 33%. Germ cell arrests are rare phenotypes and have a poor prognosis for spermatozoid recovery. Overall, histological examination (still the only way to fully describe spermatogenesis) must be qualitative and quantitative, with the adoption of a standardized, universally understood terminology. It is essential to compare the histological data with (i) recovery of testicular spermatozoids, (ii) clinical, ultrasound, hormonal and genetic data and (iii) the outcome of IVF/ICSI procedures.

A differential cytokine expression profile is induced by highly purified human menopausal gonadotropin and recombinant follicle-stimulating hormone in a pre- and postovulatory mouse follicle culture model

To compare the differential effects of highly purified (HP) hMG or recombinant FSH (rFSH) on cytokine expression before and after ovulation in an in vitro mouse ovarian follicle model. A prospective laboratory in vitro study. A university-based reproductive biology laboratory. MATERIAL(S): Mechanically isolated mouse preantral follicles from 14-day-old prepubertal mouse ovaries (F1 hybrids: C57BL/6JxCBA/ca). Randomly distributed mouse early preantral follicles were exposed to two hyperstimulation conditions with either HP-hMG or rFSH. An ovulatory stimulus was given using hCG/epidermal growth factor. Conditioned media from the two culture conditions were collected on the days before and after in vitro ovulation. Conditioned media were compared for their relative cytokine profile content as measured by a cytokine antibody array analysis. Relative concentrations of 62 cytokines in conditioned media before and after ovulation. Statistically significant increase in the production of a number of cytokines was found after HP-hMG stimulation compared with rFSH: 14 and 24 pre- and post-rhCG, respectively. Cytokines with the largest significant difference (more than 5 times) before and after ovulation included thymus-expressed cytokine (TECK), sTNFRI, and SDF-1alpha. The cytokines that are most strongly related to oocyte and embryo quality and implantation and that have been related to oocyte yield and maturation were significantly higher with HP-hMG. The significant differences in follicular cytokine production induced by HP-HMG and rFSH before and after in vitro ovulation might explain the difference in treatment outcome.

Differential effects of insulin-like growth factor-I and gonadotropins on the proliferative activity of two subgroups of granulosa cells: cumulus oophorus and mural granulosa cells

Objective: To evaluate the physiological role of insulin-like growth factor-I (IGFI) and its interaction with gonadotropins in cell replication of two types of granulosa cells (cumulus oophorus, CC) and mural granulosa cells (MGC). Design: Controlled randomized study of the action and interaction of gonadotropins and IGFI on granulosa cell replication in the rat. Setting: A university reproductive biology laboratory. Intervention(s): The study examined the in vivo treatment with gonadotropins or an analogue of IGFI, long Arg 3-insulin-like growth factor (LR 3-IGFI), which does not bind to IGFI-binding proteins. Main Outcome Measure(s): Granulosa cell replication was evaluated by the use of 3H-thymidine incorporation. Result(s): It was noted that the CC replicate much faster than the MGC. These two types of granulosa cells have very different dose response curves to IGFI. Differential responses were seen in animal cells exposed to long Arg 3-insulin-like growth factor in vivo and then exposed to FSH and LH in vitro. Conclusion(s): Although murine granulosa cells show proliferative activity when they are exposed to IGFI, the two types, CC and MGC, respond differently. IGFI is not the sole mediator of the action of FSH, and these two chemicals may act independently or in concert.

Immunofluorescent evidence of the platelet-activating factor receptor on human spermatozoa

Objective: To determine the presence of the platelet-activating factor (PAF) receptor on human spermatozoa. Design: Prospective analysis. Setting: University-based reproductive biology laboratory. Patient(s): Spermatozoa obtained from men undergoing routine semen analysis. Intervention(s): Spermatozoa (n = 10) were exposed to PAF, sheep anti-PAF antibody, and fluorescein isothiocyanate–conjugated rabbit antisheep antibody, and then evaluated by fluorescent microscopy. Main Outcome Measure(s): Assessment of fluorescent intensity at four locations on the spermatozoa (tail, midpiece, proximal head, and acrosome region). Result(s): Immunofluorescence demonstrated the presence of the PAF receptor on human spermatozoa. The PAF receptor was most prevalent at two sites on the spermatozoa: the midpiece and the proximal head. Conclusion(s): The PAF receptor is present on human spermatozoa. Platelet-activating factor may affect the motility of spermatozoa through a receptor-mediated mechanism at the midpiece and/or proximal head.

Legislative efforts affecting the reproductive biology laboratory

Legislative initiatives recently enacted in the USA will affect daily operations in the reproductive biology laboratory. These regulations affect laboratory safety, staffing, quality assurance, and record keeping. They mandate laboratory accreditation including certification, inspection, and proficiency testing. Laboratory standards of practice and costs of operation may be increased with mandatory compliance.