In vitro sperm generation from immature mouse testicular tissue using plasma rich in growth factors

Abstract

Background

Culture medium enriched with Knockout serum replacement (KSR) can produce in vitro mouse sperm, but it is inefficient, strain-specific and contains bovine products, which limits its use in the human clinic. The study aimed to optimize the culture medium for testicular tissue by using plasma rich in growth factors (PRGF) as a serum supplement, addressing the limitations of KSR.

Methods

Immature testicular tissues from NMRI mice were cultured for 14 days to identify the optimal PRGF concentration using histological analysis and tubular integrity scoring. Subsequently, tissues were cultured for 42 days with the optimal PRGF concentration and compared to a control group with 10% KSR, followed by evaluation through histological, tubular integrity, and immunofluorescence assays.

Results

After 14 days, 5% PRGF media significantly preserved tubule integrity better than 10% and 20% PRGF, performing similarly to 10% KSR. However, after 42 days, the integrity scoring revealed significantly a higher percentage of well-preserved tubules in 5% PRGF compared to 10% KSR. Additionally, only PRGF supported spermatogenesis to the production of flagellated sperm. Real-time PCR analysis revealed that transcript levels of Plzf, Tekt1, and Tnp1 were significantly elevated in 5% PRGF compared to 10% KSR. Immunofluorescence and quantitative analysis confirmed enhanced spermatogenesis progression in 5% PRGF media, with significantly increased numbers of PLZF + spermatogonia, SYCP3 + spermatocytes, ACRBP + spermatids, and Ki67 + proliferating cells per tubule compared to 10% KSR. Moreover, 5% PRGF showed a significantly lower mean fluorescence intensity of the pro-apoptotic marker Bax, with no significant difference in the anti-apoptotic marker Bcl-2 compared to KSR.

Conclusions

The findings suggest that 5%PRGF is a viable alternative to KSR in mouse testicular tissue cultures, promoting structural integrity and spermatogenesis up to the production of flagellated sperm. The results highlight PRGF’s potential to improve culture media for in vitro sperm production, suggesting promising avenues for future human research.

Graphical Abstract

Background

Male infertility is a significant factor contributing to infertility in couples, particularly observed in patients with non-obstructive azoospermia (NOA) and prepubertal boys undergoing cancer treatments such as chemotherapy and radiotherapy [1]. Consequently, a critical objective of reproductive medicine is to induce and resume in vitro spermatogenesis using these patients’ spermatogonial stem cells, aiming to produce functional sperm for conception through assisted reproductive technologies (ART) [2].

The first report of in vitro spermatogenesis dates back to 1920, involving the culture of rabbit testicular pieces on rabbit plasma for several days. After nine days, primary leptotene spermatocytes were observed [3]. In 1937, pachytene prophase meiosis I spermatocytes were produced by culturing immature mouse testicular tissue on a clot containing equal parts fowl plasma and fowl embryo extract [4]. About two decades later, Trowell developed the gold standard culturing system known as the gas-liquid interphase method [5].

Since then, many advancements have been made in in vitro spermatogenesis studies. In 2003, haploid spermatids were obtained by culturing testicular tissue from 5-day-old mice for two weeks in media containing Dulbecco’s Modified Eagle Medium (DMEM) and 10% Fetal Bovine Serum (FBS). Although haploid round spermatids were successfully injected into oocytes, the eggs did not develop past the eight-cell stage [6].

To overcome these limitations, Sato et al. used KnockOut Replacement Serum (KSR) as a supplement in the testis culture medium. This study was the first to obtain both functional round spermatids and sperm from testicular tissue cultures of C57BL/6 transgenic mice [7].

Although culture media containing 10% KSR can stimulate in vitro spermatogenesis in immature mouse testicular tissue, several significant issues remain. Firstly, the use of KSR leads to occasional sperm formation, meaning the efficiency of in vitro spermatogenesis does not match that of in vivo spermatogenesis [8, 9]. Secondly, KSR is ineffective for other mouse strains and species, and it can only fully induce in vitro spermatogenesis in testicular tissue from neonatal C57BL/6J mice [10]. Therefore, developing suitable culture media for different strains and species is crucial. Thirdly, the KSR-containing medium is derived from bovine serum and contains xenobiotic products, limiting its application to human clinics [9]. As a result, media supplementation used to induce spermatogenesis in testicular tissue culture must be optimized.

In recent years, plasma rich in growth factors (PRGF) has been increasingly used as a source of growth factors in animal and clinical studies [11,12,13,14]. PRGF is a subtype of platelet-rich plasma, a derivative of autologous blood consisting of high concentrations of platelets rich in growth factors, which play important roles in stimulating and accelerating tissue regeneration, as well as cell proliferation and differentiation [15]. The use of autologous PRGF is safe and cost-effective and has received clinical use authorization from the European Community and the U.S. Food and Drug Administration [16].

Therefore, these advantages of PRGF present new opportunities for exploring its application as a serum supplement in culture media to induce in vitro spermatogenesis in immature mouse testicular tissue.

Materials and methods

Ethics

Five-day postpartum (dpp) male NMRI (Naval Medical Research Institute) mice were housed in air-conditioned rooms maintained at a constant temperature of 22 ± 2 °C and 55 ± 5% humidity, with a 12-hour light/dark cycle. This study was conducted in accordance with the guidelines outlined in the Iranian Guide for the Care and Use of Laboratory Animals (2020) and was approved by the Ethical Committee of the Faculty of Medical Sciences at Tarbiat Modares University, with permission number IR.MODARES.REC.1399.043. The work has been reported in line with the ARRIVE guidelines 2.0.

Culture media and reagents

The basal culture medium used was α-Minimum Essential Medium (α-MEM) (Invitrogen), supplemented with 10% KSR and various concentrations of PRGF. Additionally, 100 IU/mL penicillin/streptomycin and 50 µg/mL gentamicin (Invitrogen, Carlsbad, CA, USA) were added to the culture medium of both KSR and PRGF groups.

Determining the optimized concentration of PRGF by short-term testicular culture (14 days)

During the initial phase of this investigation, testicular tissues were subjected to a 14-day culture period in basal media supplemented with varying concentrations of PRGF (5%, 10%, and 20%) and a control group with 10% KSR. Subsequently, to identify the most effective PRGF concentration, a thorough histological analysis and an assessment of tubular structure integrity were conducted (Fig. 1A-D).

Fig. 1

The experimental design consisted of several steps. (A) First, PRGF was prepared from the blood of adult male donors, and (B) immature mouse testis tissue was extracted. (C) The testicular tissue was then cultured for 14 days to determine the optimal dose of PRGF. (D) The cultured tissues were histologically evaluated using hematoxylin and eosin staining. (E Next, the testicular tissue was cultured for 42 days using the determined optimal dose of PRGF. After the 42-day culture, the tissues were fixed for histological analysis using hematoxylin and eosin staining (F) and further analyzed using immunofluorescence techniques (G)

Evaluating the efficiency of optimal PRGF concentration in long-term testicular culture (42 days)

In the subsequent phase of the study, testicular tissues were cultured for 42 days in basal media enriched with the previously determined optimal concentration of PRGF and, for comparison, in media containing 10% KSR as a control. Following this extended culture period, a comparative histological examination, an evaluation of tubular integrity, and an immunofluorescence assay were performed to assess the performance of the optimized PRGF-supplemented media relative to the control (Fig. 1E-G).

PRGF preparation

To prepare PRGF, 300 mL of venous blood was obtained from six adult male donors using 50 mL blood extraction tubes containing 3.8% sodium citrate as an anticoagulant. Each sample was centrifuged at 2000 g for 4 min at room temperature (RT), resulting in three layers: red blood cells, white blood cells, and plasma. The plasma was subsequently separated and subjected to a second centrifugation at 5000 g for a duration of 5 min at RT. The plasma was then divided into two equal fractions, F1 and F2. The upper portion, F1, contained several platelets similar to those found in peripheral blood, while fraction F2 consisted of the upper layer of the buffy coat, containing the highest concentration of platelets. The F2 fraction was selected for activation and pooled to minimize variability in platelet lysis caused by variations in F2 growth factor concentrations across donors [17]. To activate the platelets, 10% calcium chloride (50 µl/mL of plasma) was added, and the falcon tube was placed in a water bath at 39 °C for 2 h. A gelatinous layer of blood cells formed gradually (Fig. 2A-B). The liquid surrounding the gelatinous layer was collected and centrifuged for 5 min at 20,000 g (Fig. 2C). The resulting liquid was aliquoted in 1.5 cc microtubes and stored in a freezer at -80 °C until needed. When the PRGF serum was required, the microtubes were removed from the freezer, thawed, and centrifuged at 13,000 g for 10 min at 4 °C to prevent culture media gelation. The final product was a plasma containing activated platelets, which was then added to the media as a serum.

Fig. 2

PRGF preparation and testicular tissue culture method. (A-B) After activating fraction 2 of the plasma with 10% calcium chloride, a gelatinous layer formed. (C) The liquid surrounding this layer collected and centrifuged. (D) Tissue pieces were placed on agarose gels that were soaked with media up to four-fifths of the height

Testicular tissue culture method

After euthanizing mice by administering carbon dioxide for less than 5 min, followed by cervical dislocation, the immature testes were sectioned into 2–3 fragments, each measuring between 1 and 2 mm in diameter. As illustrated in Fig. 2-D, these tissue fragments were placed on stands made from agarose gel, and positioned within a 6-well plate. To fabricate the agarose gel, agarose powder (Karl Roth, Germany) was dissolved in distilled water at a concentration of 1.5% (w/v). This mixture was heated and then poured into a 10 cm dish. Once the gel had cooled, it was sliced into hexahedral blocks, each approximately 10 × 10 × 5 mm in size. These blocks were then immersed in culture media for a duration of 24 h or longer, allowing the media to fully replace the water content. Each block of agarose gel was designated to hold between one to four pieces of the testicular tissue. The media volume was adjusted to fill the space to four-fifths of the gel’s height. The media was refreshed every two days. The culture conditions were maintained in an incubator enriched with 5% CO₂ at a temperature of 34 °C.

Histological examination

Testicular tissues cultured for 14 and 42 days were fixed in Bouin’s fixative for 4 h at RT and subsequently embedded in paraffin. Serial Sect. (5 μm) were prepared from each specimen and stained with hematoxylin and eosin (H&E) for morphological analysis.

Quantitative real-time PCR

Total RNA was extracted from four pieces of cultured testis tissue, lasting 42 days for each group. The extraction was performed using RNX-Plus™ (Cinnagen, Iran) and following the manufacturer’s instructions. The concentration of the extracted RNA was measured using an ultraviolet spectrophotometer (DPI-l, Qiagen, IRI). DNase-treated RNA samples were then reverse transcribed into cDNA using the Revert Aid™ (Fermentase, Lithuania) first-strand cDNA synthesis kit and Oligo (dT) primers.

In preparation for the polymerase chain reaction (PCR) procedures, primers targeting specific genes—namely Plzf (spermatogonia), Tekt1 (spermatocyte), Tnp1 (spermatid), and ki67 (proliferation) were meticulously designed using an online primer design tool accessible through the NCBI website (www.ncbi.nlm.nih.gov/tools/primer-blast/) and were subsequently produced by a commercial entity (CinnaGen, Iran) (Table 1).

Table 1 Primers sequences designed for real-time PCR

Quantitative Real-time PCR (qRT-PCR) experiments were conducted utilizing Master Mix supplemented with SYBR Green I dye (Fluka, Switzerland), and the reactions were carried out in a Biosystems StepOne™ instrument (Applied Biosystems, UK).

The PCR protocol commenced with a preliminary denaturation step at 94 °C for a duration of 4 min, which was essential for the activation of the polymerase enzyme. This was followed by 40 consecutive cycles, each comprising a denaturation phase (20 s at 94 °C), an annealing phase (30 s at 57 °C), and an extension phase (20 s at 72 °C).

Upon the conclusion of the PCR run, the integrity and specificity of the reactions were verified through the examination of melting curve profiles. For each analyzed sample, both the reference gene (β-actin) and the gene of interest were concurrently amplified within the same reaction. The relative quantification of the target genes, normalized against a housekeeping gene (β-actin), was ascertained employing the comparative cycle threshold (CT) method, also known as the 2^−∆∆CT method. The expression level of each gene in the control samples, which consisted of mature mouse testis tissue, was assigned a baseline value of 1. This baseline was then utilized to compute the relative changes in expression, or fold change, for the target genes across different experimental conditions. A p-value threshold of less than 0.05 was established to determine statistical significance in the observed gene expression differences.

Dissociation of testicular tissues

For sperm detection in 42-day cultured tissues, mechanical dissociation using an insulin needle on slides was performed to release cells into phosphate-buffered saline (PBS).

Immunofluorescence staining

Tissues cultured for 42 days were fixed with 4% paraformaldehyde in PBS at 4 °C overnight. To target spermatogonial stem cells, leptotene-stage spermatocytes, and sperm-like cells, primary antibodies against promyelocytic leukemia zinc finger protein (PLZF), synaptonemal complex protein 3 (SYCP-3), and acrosin binding protein (ACRBP) were utilized. Additionally, the expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, and proliferation marker Ki67 was assessed.

After deparaffinization and rehydration, sections were treated with 10 mM trisodium citrate dihydrate (pH 6.0) in a microwave for 20 min at medium power (600 W) for antigen retrieval. Slides were then washed in TBS plus 0.03% Triton X-100 (Sigma- Aldrich) two times and nonspecific binding sites were blocked with a blocking buffer containing a 1% BSA solution in TBS for 2 h at RT. Following this, slides were washed for 3 min and incubated overnight at 4 °C with primary antibodies PLZF, SYCP-3, ACRBP, Bax, Bcl-2 (1:100, Santa Cruz Biotechnology, U.S.A.), and Ki67 (1:100, Elabscience, U.S.A.). After washing, slides were incubated with Cy3-conjugated Goat anti-Mouse IgG (1:100, Elabscience, U.S.A.) for 1 h at RT in the dark. After washing, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1:200, Sigma, Germany) for 5 min. Sections were then dehydrated through ascending ethanol concentrations, cleared with xylene, and mounted with coverslips.

Stained samples were visualized using a light and fluorescent microscope (Olympus BX50, Germany). Five random images per sample (magnification: x400) were captured and analyzed for fluorescence intensity using Image-J software (National Institutes of Health, Bethesda, MD, USA). For PLZF, SYCP-3, ACRBP, and Ki67, the mean number of antibody-positive cells per seminiferous tubule was calculated. For Bax and Bcl-2, mean fluorescence intensity was quantified following the method described by Leite et al. [18].

Integrity of seminiferous tubules

A double-blind assessment was conducted to evaluate the integrity of seminiferous tubules using semi-quantitative analysis under a light microscope at ×400 magnification, following the scoring method described by de Michele et al. [19]. For this purpose, at least 200 round or nearly round tubules were randomly selected from 15 fields in 5 histological sections for each time point and culture medium. The four parameters investigated included: the adhesion of cells to the basement membrane, cell cohesion, proportion of pyknotic nuclei (less than 5% of the total), and the clear distinction between germ cells and Sertoli cells. The scoring system for seminiferous tubule integrity ranged from 1 (poorly preserved), 2 (fair), 3 (good) to 4 (best preserved).

Statistical analysis

Graphical and statistical analyses were conducted using GraphPad Prism 4 software (GraphPad Software Inc., La Jolla, CA, USA). Quantitative data are presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA and T-tests. A significance level of P < 0.05 was considered statistically significant.

Results

Histological and tubular integrity evaluation to determine optimal PRGF concentration

Histological evaluation after 14 days of culture showed central necrosis and degeneration of seminiferous tubules in all media (Fig. 3). Furthermore, the peripheral tubules of tissues cultured in 20% and 10% PRGF media exhibited poor preservation, with large clusters of apoptotic cells. In contrast, in 10% KSR and 5% PRGF media, a torus-shaped zone with intact and well-preserved tubules surrounded the central necrotic area, and spermatogenesis was active in this zone.

Fig. 3

Histological assessment of testicular tissue from 5 dpp NMRI mice cultured for 14 days. (A) H&E staining of cultured tissues in media supplemented with 20% PRGF, (B) 10% PRGF, (C) 5% PRGF, and (D) 10% KSR. Higher magnifications of A, B, C, and D are shown in a, b, c, and d, respectively. Black and red arrowheads indicate round and elongated spermatids, respectively

According to the integrity scoring system (Fig. 4A-D), the peripheral tubules in 20% and 10% PRGF media showed a disorganized pattern, with decreased cell cohesion and adhesion to the basement membrane, and an increased number of pyknotic cells. This resulted in a significantly higher percentage of tubules with a poorly preserved integrity score (1) compared to the 5% PRGF and 10% KSR media (Fig. 4E). The percentage of tubules with a fair integrity score (2) significantly increased in media containing 20% and 10% PRGF compared to the culture group with 10% KSR. However, the percentage of tubules with good and best-preserved integrity scores (3–4) significantly increased in cultures supplemented with 5% PRGF and 10% KSR compared to 20% and 10% PRGF.

Fig. 4

The assessment of the seminiferous tubules’ integrity in cultured testes tissues over a 14-day period in media with 10% KSR and varying concentrations of PRGF (5%, 10%, and 20%). (A-D) Four distinct instances of tubules were presented, ranging from the 1 to 4 score. (A) Tubules with a score of 1 (poorly preserved) exhibited signs of poor preservation, characterized by cell necrosis in the middle of the tubule and a lack cell cohesion and adherence to the basement membrane. (B) Tubules graded as score 2, or fair, displayed pyknotic nuclei within the lumen, along with difficulty in distinction between germ cells and Sertoli cells. (C) Tubules scoring 3, or good, also contained pyknotic nuclei in the lumen. (D) The most well-preserved tubules, graded as score 4, displayed excellent cell cohesion and adherence to the basement membrane, as well as clear distinction between Sertoli cells and germ cells. (E) Integrity scoring system showed that there was no significant difference in the tubule scores between the 5% PRGF and 10% groups. However, a significant difference was observed in the percentage of tubules with scores of 1, 3, and 4 between the 5% PRGF and 10% KSR groups compared to the 10% and 20% PRGF groups. The data are presented as mean ± (SD) for three replicate experiments. The statistical significance is as follows: ****; P < 0.0001 for 5% PRGF versus 10 and 20% PRGF. ####; P < 0.0001 for KSR versus 10 and 20% PRGF. ###; P < 0.001 for KSR versus 10 and 20% PRGF. ##; P < 0.01 for KSR versus 10 and 20% PRGF

Therefore, the 5% PRGF media significantly preserved the integrity of tubules after 14 days of culture compared to 10% and 20% PRGF. No significant difference was observed in the integrity score between the 10% KSR and 5% PRGF groups throughout the 14-day culture period.

Histological and tubular integrity evaluation: assessing the efficiency of 5% PRGF media for long-term culture

Comparative histological examination of 10% KSR and 5% PRGF media after 42 days of culture revealed necrosis and degeneration of tubules in the middle part of each tissue (Fig. 5A-B). However, the 5% PRGF media supported well-preserved peripheral tubules, with spermatogonial stem cells progressing to the meiotic phase and differentiating into round and elongated spermatids. In contrast, the 10% KSR media showed poorly preserved peripheral tubules with decreased cell cohesion and adhesion to the basement membrane.

Fig. 5

Evaluation of cultured testis tissues for 42 days. (A) In the presence of 5% PRGF, the central region of the tubules exhibited degeneration. However, the peripheral tubules maintained their integrity, the spermatogonial stem cells differentiated into elongated spermatid cells. Green, blue, black and red arrow heads are spermatogonial, spermatocyte, round spermatid and elongated spermatid cells, respectively. (B) Conversely, when cultured with 10% KSR, the testicular tissue fragments showed degeneration in both the peripheral and central areas of the tubules. (C) There was a significant difference in the percentages of tubules with scores of 1, 2, 3, and 4 between the 10% KSR and 5% PRGF media after 42 days of culture. The data presented are the mean ± SD from three replicate experiments. ###; P < 0.001 for KSR versus 5% PRGF. ##; P < 0.01 for KSR versus 5% PRGF. #; P < 0.05 for KSR versus 5% PRGF. (D) Flagellated sperm were observed after mechanically dissociated tissues cultured in media containing 5% PRGF. (E) Relative expression of Plzf, Tekt1, Tnp1, and Ki67 genes. The statistical significance is as follows: ****; P < 0.0001 for control group (adult mouse testes) versus 5% PRGF and 10%KSR. ***; P < 0.001 for control group versus 5% PRGF and 10%KSR. ###; P < 0.001 for 5% PRGF versus 10% KSR

Based on the integrity scoring system (Fig. 5C), it was found that after 42 days of tissue culture, the percentage of tubules with good and best-preserved integrity scores significantly increased in the presence of 5% PRGF compared to 10% KSR. Conversely, the use of KSR-supplemented media led to a higher percentage of tubules with poor and fair integrity scores (1–2) compared to 5% PRGF. In conclusion, media containing 5% PRGF proved to be more effective in preserving the integrity of tubules after 42 days of culture when compared to 10% KSR.

After 42 days of culture, four dissociated tissues from the 10% KSR and 5% PRGF media were observed under a light microscope. Four flagellated sperm cells were observed only in the 5% PRGF media (Fig. 5D).

Expression of genes involved in spermatogenesis and proliferation

Real-time PCR analysis revealed that the transcript levels of Plzf, Tekt1, and Tnp1 were significantly elevated in tissues treated with 5% PRGF relative to those treated with 10% KSR (p < 0.05). In contrast, the expression of Ki67 remained relatively unchanged between the two treatment groups. Following culture of testicular tissues from both groups, the expression levels of the Tekt1, Tnp1, and Ki67 genes showed a significant reduction when compared to the control group, which consisted of adult mouse testes (p < 0.05) (Fig. 5E).

Assessment of immunofluorescence staining following 42 days of testicular tissue culture

Quantitative immunofluorescence results showed that the mean number of PLZF + cells (spermatogonial stem cells) per tubule in the 5% PRGF media was significantly greater than in the KSR media (P = 0.0045) (Fig. 6AB).

Fig. 6

Immunofluorescence staining and quantitative evaluation of PLZF, SYCP3 and ACRBP antibodies after 42 days of testicular tissue culture in 5% PRGF and 10% KSR media. (A) Immunofluorescence staining of PLZF + spermatogonial stem cells, SYCP3 + spermatocyte cells, and ACRBP + spermatid and sperm cells (red). Yellow arrow heads are spermatid and sperm cells. Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (B) Quantitative analysis demonstrated a significant increase in the mean number of PLZF+, SYCP3 + and ACRBP + cells per tubule in tissues cultured with 5% PRGF compared to those cultured with KSR. Statistical significance indicated by (*p ≤ 0.05) (**p ≤ 0.01). Data are presented as mean ± SD

The onset of meiosis and the differentiation of spermatogonia were marked by the emergence of cells positive for SYCP3, a marker for spermatocytes, in both culture media (Fig. 6AB). However, a quantitative analysis revealed a significant increase in the number of spermatocytes per seminiferous tubule in the medium supplemented with 5% PRGF compared to the KSR-supplemented medium (P = 0.0013).

The identification of post-meiotic markers, specifically ACRBP (a spermatid and sperm marker), confirms the presence of more advanced stages of spermatogenesis. Quantitative analysis demonstrated a statistically significant increase (P = 0.0212) in the number of ACRBP-positive cells per tubule in the 5% PRGF media compared to the KSR group (Fig. 6AB). This indicates that the 5% PRGF media is more effective in promoting the progression of spermatogenesis to later stages.

The proliferation status was evaluated by Ki67 + cell analysis, which showed a significant increase in the number of Ki67 + cells per tubule in the 5% PRGF media compared to KSR (P = 0.0293) (Fig. 7AB).

Fig. 7

Immunofluorescence staining and quantitative analysis of Ki67, Bax and Bcl-2 antibodies after 42 days of testicular tissue culture in 5% PRGF and 10% KSR media. (A) Immunofluorescence staining of the proliferation marker Ki67 (red), pro-apoptotic marker Bax (green), and anti-apoptotic marker Bcl-2 (green). Nuclei were labeled with DAPI (blue). Dashed white line: basal lamina. Scale bar = 20 μm. (B) 5% PRGF culture promoted higher cell proliferation and lower apoptosis rates compared to KSR, as indicated by a significant increase in Ki67 + cells and a lower mean fluorescence intensity of Bax in 5% PRGF-cultured tissues. No significant difference was found in Bcl-2 mean fluorescence intensity between the two culture media. Statistical significance denoted by (*p ≤ 0.05) and (***; P < 0.001). Data are presented as means ± SD

Figure 7 illustrates a significant increase in the mean fluorescence intensity of the pro-apoptotic marker Bax per field, comparing the KSR group to the 5% PRGF group, after 42 days of culture (P = 0.0004). However, the mean fluorescence intensity of the anti-apoptotic marker Bcl-2 per field in media supplemented with 5% PRGF did not show a statistically significant increase compared to KSR after 42 days of culture (P = 0.2477), as demonstrated in (Fig. 7AB).

Discussion

In vitro sperm production is a complex biological process that has challenged researchers for the past hundred years. The testicular tissue culture media supplemented with KSR is far from optimal, and there is much room for improvement. Researchers have attempted to enhance media composition by formulating chemically defined mediums, but none have resulted in successful sperm production [1, 20, 21].

This study aimed to develop a media for in vitro spermatogenesis without the use of KSR. In the first step, the effects of adding three different concentrations of PRGF to the basal media were evaluated during 14 days of NMRI mice testes culture. Notably, the media supplemented with 20% and 10% PRGF exhibited poorly preserved peripheral tubules, characterized by decreased cell cohesion and attachment to the basement membrane, and an increased number of pyknotic cells. These findings suggest that high concentrations of PRGF may have detrimental effects on the structural integrity and cellular viability of the seminiferous tubules due to increased cytotoxicity. Similar to the research, various studies have indicated that using overly high platelet concentrations, due to the presence of excessive growth factors, can suppress the proliferation of osteoblasts, fibroblasts, and adipose-derived mesenchymal stem cells [22,23,24].

Conversely, media supplemented with 5% PRGF, similar to 10% KSR, maintained a greater percentage of tubules with good and best-preserved integrity (scores 3–4) compared to 20% and 10% PRGF. A separate study, consistent with the findings, demonstrated that 5% PRP media led to significantly higher viability and proliferation of spermatogonial stem cells compared to 1%, 2.5%, and 10% PRP after a 14-day culture [25].

In contrast, the evaluation of the long-term culture at 42 days revealed distinct differences between the 10% KSR and 5% PRGF groups. In 5% PRGF media, the integrity of peripheral tubules was significantly better preserved compared to 10% KSR, and spermatogenesis progressed up to the production of flagellated sperm. In addition, real-time PCR analysis demonstrated that the use of 5% PRGF significantly enhanced the transcript levels of Plzf, Tekt1, and Tnp1 in comparison to tissues treated with 10% KSR (p < 0.05).This finding suggests that 5% PRGF may be an effective substitute for KSR in supporting in vitro spermatogenesis and mature sperm production. PRGF is a human autologous blood derivative rich in proteins and growth factors such as insulin-like growth factor 1 (IGF-I), transforming growth factor-β1 (TGF-β1), epidermal growth factor (EGF), bone morphogenetic proteins (BMP), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) [26], which can play crucial roles in improving the in vitro spermatogenesis process.

In mouse testis culture, IGF-1, TGF-α, and EGF can induce differentiation of type A spermatogonial cells [27, 28]. Consequently, the increased count of PLZF-positive spermatogonial and SYCP3-positive spermatocytes observed in each tubule following a 42-day culture in 5% PRGF media, as compared to KSR, is likely due to the presence of these growth factors in PRGF.

The significant difference in the number of ACRBP + spermatid and sperm cells per tubule observed in PRGF-cultured tissues compared to KSR can likely be attributed to the presence of TGF-β in PRGF. This is because TGF-β plays a key role in the auto/paracrine pathways that regulate the meiotic differentiation of spermatocytes [29]. Additionally, IGF-1 supplementation in the media has been demonstrated to enhance the quantity of both round and elongated spermatids in cultured mouse testicular tissue [30].

Growth factors such as BMP2, BMP7, HGF, IGF-1, and VEGF play a stimulatory role in the proliferation of spermatogonia and Sertoli cells during in vitro spermatogenesis [31,32,33]. The presence of these factors can effectively contribute to a significant increase in the number of Ki67 + cells in tissues cultured with PRGF.

The application of 5% PRGF significantly downregulated Bax expression while upregulating Bcl-2 expression, suggesting its anti-apoptotic effects. These effects can be attributed to HGF and IGF-1 within PRGF, as previous research has demonstrated their roles in promoting the survival of germ cells in rats and mice, respectively [30, 33]. Moreover, other studies have highlighted the importance of PRGF in promoting the proliferation, differentiation, and survival of stem cells under in vitro conditions [34, 35].

In contrast, KSR lacks this diverse profile of targeted growth factors and proteins, which likely limits its ability to provide the same level of support for germ cell development and survival. Thus, the combination of growth factors within PRGF acts synergistically to enhance spermatogenesis and reduce apoptosis more effectively than KSR.

The findings suggest that KSR did not successfully induce complete spermatogenesis or even maintain the structural integrity of the peripheral tubules in NMRI mice after a 42-day culture. Initial reports suggested that KSR could induce complete in vitro spermatogenesis in mice [7, 36]. However, later studies revealed that the effectiveness of KSR in successful spermatogenesis is strain-dependent and worked with C57BL/6 mice testes. Specifically, it was found that KSR was poorly supported in vitro spermatogenesis in testis of B6D2F2 mice [10]. In another study, it was observed that the basal media formulation used by Sato et al. [7] (𝛼MEM and KSR) rarely promoted spermatogonial cell progression up to the spermatocyte stage in CD1 and C57 mice strains [37]. Furthermore, current evidence suggests KSR is not sufficient to achieve complete in vitro spermatogenesis in rat and human immature testicular tissues [19, 38,39,40].

Besides the inefficiencies noted for KSR in testicular tissue culture, the limitation of its clinical application highlights the necessity for an appropriate alternative. Given these limitations, human autologous blood derivatives have been proposed as a preferable alternative to supplements derived from xenogeneic sources, such as KSR [41,42,43,44,45,46]. The use of autologous PRGF, as a personalized medicine approach with promising potential for maximum patient safety [47] and maintenance of genomic stability [24, 48], presents a suitable opportunity for future research in the field of in vitro spermatogenesis in different species.

In this study, we successfully produced flagellated spermatozoa using immature mouse testicular tissue culture. However, a notable limitation was the absence of an investigation into the fertility potential of the in vitro-generated sperm and spermatids using microinsemination techniques. Another limitation of this study is that while NMRI mice are commonly used in reproductive biology research, they may not fully capture the complexities of human reproductive physiology. Consequently, these aspects warrant further scientific inquiry.

Conclusion

In conclusion, our study presents compelling evidence that 5% PRGF is a promising substitute for KSR in in vitro spermatogenesis, enhancing structural integrity and cell viability in NMRI mouse testicular tissue cultures. Our findings show that 5% PRGF not only preserves the architecture of seminiferous tubules but also supports the progression of spermatogenesis up to the production of flagellated sperm. While our study achieved the production of mature sperm, future research should investigate the fertility potential of these spermatozoa to assess their practical applications in reproductive technologies. These results highlight the potential of autologous PRGF as a crucial element in optimizing culture media for in vitro sperm production and propose promising avenues for future research in human applications.